Charged lipids and uses for the same

ABSTRACT

The present invention is directed to charged lipids, compositions comprising charged lipids, and the use of these compositions in drug delivery, targeted drug delivery, therapeutic imaging and diagnostic imaging, as well as their use as contrast agents.

RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 08/823,791,filed Mar. 21, 1997; a continuation-in-part of U.S. application Ser. No.08/851,780 filed May 6, 1997; a continuation-in-part of U.S. applicationSer. No. 08/877,826 filed Jun. 18, 1997; and a continuation-in-part ofU.S. application Ser. No. 08/887,215 filed Jul. 2, 1997, the disclosuresof each of which are hereby incorporated by reference hirein in theirentirety.

FIELD OF THE INVENTION

The present invention is directed to charged lipids, compositionscomprising charged lipids, and the use of these compositions in drugdelivery, targeted drug delivery, therapeutic imaging and diagnosticimaging, as well as their use as contrast agents.

BACKGROUND OF THE INVENTION

To improve drug delivery, lessen toxicity and improve efficacy, variousdrug delivery systems have been devised. Drug delivery systems haveincluded liposomes which are generally composed of neutral orzwitterionic lipids. The lipids in liposomes arrange themselves intobilayers and entrap one (unilamellar) or more (oligo- or multilamellar)spaces. The spaces between the bilayers of the lipids are usually filledwith water. In conventional water filled liposomes, drugs are usuallyentrapped in the internal aqueous space, although in some cases they maybe incorporated in the wall forming materials of the lipid bilayer. Inconventional liposomes, it is often difficult to entrap a highconcentration of a drug. Relying upon the internal entrapment of thedrug, the efficiency necessarily depends upon the volume of fluidoutside of the liposomes and circumscribed within the internal aqueousvesicular space. To improve the efficiency of drug entrapment, varioustechniques, such as ionic or pH gradients, have been employed. Still,the efficiency of drug encapsulation is less than desired. On long termstorage, drugs entrapped within liposomes may leak out of the internalaqueous space into the surrounding milieu. The drug may therefore belost from its desired intra-liposomal location. This is particularlyproblematic when there is a high concentration of drug entrapped withinthe liposomes and there is an osmotic gradient across the bilayermembrane. New and better methods of entrapping drugs in liposomes wouldbe beneficial.

Studies have described the effects of calcium and other multivalentcations on membrane asymmetry, lipid distribution, vesicle size,aggregation and fusion. Although the underlying physical causes for thephenomena are debatable, general consensus exists that multivalentcations, such as calcium and magnesium, in the external environment ofphospholipid vesicles cause the structures to aggregate into larger,multilamellar structures and promotes fusion. Barium and strontium ionshave also been investigated in this regard. Duzgunes et al.,Biochemistry, 23:3486-3494 (1984). Species of phospholipids that areparticularly pronounced in these effects are the subjet ofinvestigation, as described, for example, by Leckband, et al.,Biochemistry 32:1127-1140 (1993), Tilley et al., Biogenic Amines,5:69-74 (1988) and Kwon, et al., Colloids and Surfaces B, 3:25-30(1994).

Other areas of investigation focused on the effect of calcium-inducedaggregation on phase transition temperature and whether aggregation andfusion phenomena have a temperature dependance. Duzgunes, supra, Kwon,supra, and Tilcock et al., Biochemistry, 23:2696-2703 (1984). Theeffects of calcium-induced aggregation are so pronounced that effortshave been undertaken to limit the effect in order to control the size ofliposomes used in drug delivery systems by forming vesicles in whichcalcium ions are confined to outer surfaces of the bilayer. EuropeanPatent Publication EP 579 703.

Another form of prior art has been the development of polymericmicrospheres. Polymeric microspheres may retain the drugs to a betterextent than liposomes, but there may be problems with biodegradabilityand toxicity.

The present invention is directed to, among other things, thedevelopment of new and improved drug and contrast media delivery systemsthat overcome the problems associated with the prior art.

SUMMARY OF THE INVENTION

The present invention describes methods of delivering bioactive agentsto a patient and/or treating conditions in a patient comprisingadministering to the patient a composition comprising a charged lipid, acounter ion, a lipid covalently bonded to a polymer and a bioactiveagent, and applying therapeutic ultrasound to the patient to facilitatedelivery of the bioactive agent in a desired region of the patient. Ifdesired, the methods may further comprise imaging the patient to monitorthe location of the composition. The composition may further comprise,for example, one or more of neutral lipids, charged lipids, gases,gaseous precursors, liquids, oils, diagnostic agents, targeting ligandsand/or other bioactive agents.

The present invention describes methods of delivering bioactive agentsto a patient and/or treating conditions in a patient comprisingadministering to the patient a composition comprising a charged lipid, acounter ion, a lipid covalently bonded to a polymer, a bioactive agentand a targeting ligand. If desired, the methods may further compriseimaging the patient to monitor the location of the composition and/orapplying therapeutic ultrasound to the patient to facilitate delivery ofthe bioactive agent in a desired region of the patient. The compositionmay further comprise, for example, one or more of neutral lipids,charged lipids, gases, gaseous precursors, liquids, oils, diagnosticagents and/or other bioactive agents.

The present invention also describes methods of providing an image of aninternal region of a patient comprising administering to the patient acomposition comprising a charged lipid, an counter ion, and a lipidcovalently bonded to a polymer. The methods may further comprisescanning the patient using diagnostic imaging to obtain visible imagesof the internal region of the patient. The composition may furthercomprise, for example, one or more of neutral lipids, charged lipids,gases, gaseous precursors, liquids, oils, diagnostic agents, targetingligands and/or bioactive agents.

The present invention also describes methods of diagnosing the presenceof diseased tissue in a patient comprising administering to the patienta composition comprising a charged lipid, a counter ion, and a lipidcovalently bonded to a polymer. The methods may further comprisescanning the patient using diagnostic imaging to obtain a visible imageof any diseased tissue in the patient. The composition may furthercomprise, for example, one or more of neutral lipids, charged lipids,gases, gaseous precursors, liquids, oils, diagnostic agents, targetingligands and/or bioactive agents.

In addition, the present invention describes novel contrast agentscomprising a charged lipid, a counter ion, and a lipid covalently bondedto a polymer. The contrast agents may further comprise, for example, oneor more of neutral lipids, charged lipids, gases, gaseous precursors,liquids, oils, diagnostic agents, targeting ligands and/or bioactiveagents.

These and other aspects of the invention will become apparent from thefollowing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are illustrative examples of the compositions of thepresent invention. FIG. 1A is an illutration of a composition of thepresent invention comprising a charged lipid, a counter ion and a lipidcovalently bonded to a polymer, while FIG. 1B is an illustration of acomposition of the present invention comprising a charged lipid, acounter ion, a lipid covalently bonded to a polymer and a targetingligand.

FIGS. 2A and 2B are sizing profiles of compositions prepared withvariable amounts of dimyristoylphosphatidylcholine (DMPC),dimyristoylphosphatidic acid (DMPA) anddipalmitoylphosphatidylethanolamine-polyethylene glycol 5,000(DPPE-PEG-5000) either in the absence of CaCl₂ (FIG. 2A) or with CaCl₂added after liposome formation (FIG. 2B).

FIGS. 3A and 3B are sizing profiles of compositions prepared withvariable amounts of dipalmitoylphosphatidylcholine (DPPC),dipalmitoylphosphatidic acid (DPPA) and DPPE-PEG-5000 either in theabsence of CaCl₂ (FIG. 3A) or with CaCl₂ added prior to liposomeformation (FIG. 3B).

FIGS. 4A and 4B are sizing profiles of compositions prepared withvariable amounts of DPPC, DPPA and DPPE-PEG-5000 either in the absenceof CaCl₂ (FIG. 4A) or with CaCl₂ added prior to liposome formation (FIG.4B). Unlike FIGS. 3A and 3B, FIGS. 4A and 4B have DPPA in excess ofDPPC.

DETAILED DESCRIPTION OF THE INVENTION

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

"Amphiphilic moiety" or "amphiphile" refers to a synthetic,semi-synthetic (modified natural) or naturally-occurring compound havinga water-soluble, hydrophilic portion and a water-insoluble, hydrophobicportion. Preferred amphiphilic compounds are characterized by a polarhead group, for example, a phosphatidylcholine group, and one or morenonpolar, aliphatic chains, for example, palmitoyl groups. "Fluorinatedamphiphilic moiety" refers to an amphiphilic compound in which at leastone hydrogen atom of the amphiphilic compound is replaced with afluorine atom. In a preferred form, the fluorinated amphiphiliccompounds are polyfluorinated. "Polyfluorinated amphiphilic moiety"refers to amphiphilic compounds which contain two or more fluorineatoms. "Perfluorinated amphiphilic moiety" refers to amphiphiliccompounds in which all the hydrogen atoms have been replaced with afluorine atom. "Amphipathy" refers to the simultaneous attraction andrepulsion in a single molecule or ion containing one or more groupshaving an affinity for the phase or medium in which they are dissolved,emulsified and/or suspended, together with one or more groups that tendto be expelled from the involved phase or medium.

"Lipid" refers to a naturally-occurring, synthetic or semi-synthetic(modified natural) compound which is generally amphipathic. Lipidstypically comp rise a hydrophilic component and a hydrophobic component.Exemplary lipids include, foir example, fatty acids, neutral fats,phosphatides, fluorinated lipids, oils, fluorinated oils, glycolipids,surface active agents (surfactants and fluorosurfactants), aliphaticalcohols, waxes, terpenes and steroids. The phrase semi-synthetic(modified natural) denotes a natural compound that has been chemicallymodified in some fashion. Lipids are also referred to herein as"stabilizing materials" or "stabilizing compounds." A "fluorinatedlipid" refers to a lipid in which at least one hydrogen atom of thelipid is replaced with a fluorine atom.

"Surfactant" refers to a surface active agent, which is a compound thatalters surface tension. Surface active agents include, for example,detergents, wetting agents and emulsifiers. "Fluorosurfactant" refers toa surfactant in which at least one hydrogen atom of the surfactant isreplaced with a fluorine atom.

"Vesicle" refers to an entity which is generally characterized by thepresence of one or more walls or membranes which form one or moreinternal voids. Vesicles may be formulated from a stabilizing materialsuch as a lipid, including the various lipids described herein. Thelipids may be natural, synthetic or semi-synthetic. The walls ormembranes may be concentric or otherwise. The lipids may be in the formof one or more monolayers or bilayers. In the case of more than onemonolayer or bilayer, the monolayers or bilayers may be concentric.Stabilizing compounds may be used to form a unilamellar vesicle(comprised of one monolayer or bilayer), an oligolamellar vesicle(comprised of about two or about three monolayers or bilayers) or amultilamellar vesicle (comprised of about three or more layers orcomprised of about three or more monolayers or bilayers). The walls ormembranes of vesicles may be substantially solid (uniform), or they maybe porous or semi-porous. The vesicles described herein inchlde and mayalso be referred to as, for example, cochleates, liposomes, micelles,bubbles, microbubbles, microspheres, lipid-coated bubbles, nanospheres,microballoons, microcapsules, aerogels, clathrate bound vesicles,hexagonal H II phase structures, and the like. The internal void of thevesicles may optionally be filled with water, oil, liquids, gases,gaseous precursors, bioactive agents, diagnostic agents, and/or othermaterials.

"Liposome" refers to a generally spherical or spheroidal cluster oraggregate of amphipathic compounds, including lipid compounds, typicallyin the form of one or more concentric layers, for example, monolayers,bilayers or multi-layers. They may also be referred to herein as lipidvesicles. The liposomes may be formulated, for example, from ioniclipids and/or non-ionic lipids. Liposomes formulated from non-ioniclipids may be referred to as niosomes. Liposomes formulated, at least inpart, from cationic lipids or anionic lipids may be referred to ascochleates.

"Cochleate" generally refers to a multilamellar lipid vesicle that isgenerally in the shape of a spiral or a tubule.

"Micelle" refers to colloidal entities formulated from lipids. Micellesmay comprise a monolayer, bilayer, or hexagonal H II phase structure.

"Aerogel" refers to generally spherical or spheroidal entities which arecharacterized by a plurality of small internal voids. The aerogels maybe formulated from synthetic materials (e.g., a foam prepared frombaking resorcinol and formaldehyde) and/or natural materials, such ascarbohydrates (polysaccharides) or proteins.

"Clathrate" refers to a solid, semi-porous or porous particle which maybe associated with vesicles. Preferably, clathrates form a cage-likestructure containing cavities which comprise one or more vesicles boundto the clathrate. A stabilizing material may be associated with theclathrate to promote the association of the vesicle with the clathrate.Clathrates may be formulated from, for example, porous apatites, such ascalcium hydroxyapatite, and precipitates of polymers and metal ions,such as alginic acid precipitated with calcium salts.

"Emulsion" refers to a mixture of two or more generally immiscibleliquids, and is generally in the form of a colloid. The mixture may beof lipids, for example, which may be homogeneously or heterogeneouslydispersed throughout the emulsion. Alternatively, the lipids may beaggregated in the form of, for example, clusters, layers or tubules,including monolayers, bilayers or multi-layers.

"Suspension" or "dispersion" refers to a mixture, preferably finelydivided, of two or more phases (solid, liquid or gas), such as, forexample, liquid in liquid, solid in solid, gas in liquid, and the likewhich preferably can remain stable for extended periods of time.

"Hexagonal H II phase structure" generally refers to a tubularaggregation of lipids in liquid media, for example, aqueous media, inwhich the hydrophilic portion(s) of the lipids generally face inwardlyin association with an aqueous liquid environment inside the tube. Thehydrophobic portion(s) of the lipids generally radiate outwardly and thecomplex assumes the shape of a hexagonal tube. A plurality of tubes isgenerally packed together in the hexagonal phase structure.

"Gas filled vesicle" refers to a vesicle having a gas encapsulatedtherein. "Gaseous precursor filled vesicle" refers to a vesicle having agaseous precursor encapsulated therein. The vesicles may be minimally,partially, substantially, or completely filled with the gas and/orgaseous precursor. The term "substantially" as used in reference to thegas and/or gaseous precursor filled vesicles means that greater thanabout 30% of the internal void of the substantially filled vesiclescomprises a gas and/or gaseous precursor. In certain embodiments,greater than about 40% of the internal void of the substantially filledvesicles comprises a gas and/or gaseous precursor, with greater thanabout 50% being more preferred. More preferably, greater than about 60%of the internal void of the substantially filled vesicles comprises agas and/or gaseous precursor, with greater than about 70% or 75% beingmore preferred. Even more preferably, greater than about 80% of theinternal void of the substantially filled vesicles comprises a gasand/or gaseous precursor, with greater than about 85% or 90% being stillmore preferred. In particularly preferred embodiments, greater thanabout 95% of the internal void of the vesicles comprises a gas and/orgaseous precursor, with about 100% being especially preferred.Alternatively, the vesicles may contain no or substantially no gas orgaseous precursor.

"Patient" refers to animals, including mammals, preferably humans.

"Region of a patient" refers to a particular area or portion of thepatient and in some instances to regions throughout the entire patient.Exemplary of such regions are the pulmonary region, the gastrointestinalregion, the cardiovascular region (including, myocardial tissue), therenal region as well as other bodily regions, tissues, lymphocytes,receptors, organs and the like, including the vasculature andcirculatory system, and as well as diseased tissue, including canceroustissue. "Region of a patient" includes, for example, regions to beimaged with diagnostic imaging, regions to be treated with a bioactiveagent, regions to be targeted for the delivery of a bioactive agent, andregions of elevated temperature. The "region of a patient" is preferablyinternal, although, if desired, it may be external. The phrase"vasculature" denotes blood vessels (including arteries, veins and thelike). The phrase "gastrointestinal region" includes the region definedby the esophagus, stomach, small and large intestines, and rectum. Thephrase "renal region" denotes the region defined by the kidney and thevasculature that leads directly to and from the kidney, and includes theabdominal aorta.

"Bioactive agent" refers to a substance which may be used in connectionwith an application that is therapeutic or diagnostic, such as, forexample, in methods for diagnosing the presence or absence of a diseasein a patient and/or methods for the treatment of a disease in a patient."Bioactive agent" refers to substances which are capable of exerting abiological effect in vitro and/or in vivo. The bioactive agents may beneutral, positively or negatively charged. Suitable bioactive agentsinclude, for example, prodrugs, diagnostic agents, therapeutic agents,pharmaceutical agents, drugs, oxygen delivery agents, blood substitutes,synthetic organic molecules, proteins, peptides, vitamins, steroids,steroid analogs and genetic material, including nucleosides, nucleotidesand polynucleotides.

"Diagnostic agent" refers to any agent which may be used in connectionwith methods for imaging an internal region of a patient and/ordiagnosing the presence or absence of a disease in a patient. Diagnosticagents include, for example, contrast agents for use in connection withultrasound imaging, magnetic resonance imaging (MRI), nuclear magneticresonance (NMR), computed tomography (CT), electron spin resonance(ESR), nuclear medical imaging, optical imaging, elastography,radiofrequency (RF) and microwave laser. Diagnostic agents may alsoinclude any other agents useful in facilitating diagnosis of a diseaseor other condition in a patient, whether or not imaging methodology isemployed. As defined herein, a "diagnostic agent" is a type of bioactiveagent.

"Therapeutic agent," "pharmaceutical agent" or "drug" refers to anytherapeutic or prophylactic agent which may be used in the treatment(including the prevention, diagnosis, alleviation, or cure) of a malady,affliction, condition, disease or injury in a patient. Therapeuticallyuseful genetic materials, peptides, polypeptides and polynucleotides maybe included within the meaning of the term pharmaceutical or drug. Asdefined herein, a "therapeutic agent," "pharmaceutical agent" or "drug"is a type of bioactive agent.

"Targeting ligand" refers to any material or substance which may promotetargeting of tissues and/or receptors in vivo or in vitro with thecompositions of the present invention. The targeting ligand may besynthetic, semi-synthetic, or naturally-occurring. Materials orsubstances which may serve as targeting ligands include, for example,proteins, including antibodies, antibody fragments, hormones, hormoneanalogues, glycoproteins and lectins, peptides, polypeptides, aminoacids, sugars, saccharides, including monosaccharides andpolysaccbarides, carbohydrates, vitamins, steroids, steroid analogs,hormones, cofactors, and genetic material, including nucleosides,nucleotidies, nucleotide acid constructs and polynucleotides. A"precursor" to a targeting ligand refers to any material or substancewhich may be converted to a targeting ligand. Such conversion mayinvolve, for example, anchoring a precursor to a targeting ligand.Exemplary targeting precursor moieties include maleimide groups,disulfide groups, such as ortho-pyridyl disulfide, vinylsulfone groups,azide groups, and α-iodo acetyl groups.

"Genetic material" refers to nucleotides and polynucleotides, includingdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The geneticmaterial may be made by synthetic chemical methodology known to one ofordinary skill in the art, or by the use of recombinant technology, orby a combination thereof The DNA and RNA may optionally compriseunnatural nucleotides and may be single or double stranded. "Geneticmaterial" also refers to sense and anti-sense DNA and RNA, which arenucleotide sequences which are complementary to specific sequences ofnucleotides in DNA and/or RNA.

"Stabilizing material" or "stabilizing compound" refers to any materialwhich is capable of improving the stability of compositions containingthe gases, gaseous precursors, bioactive agents, targeting ligands,and/or other materials described herein, including, for example,mixtures, suspensions, emulsions, dispersions, vesicles, and the like.Encompassed in the definition of "stabilizing material" are certain ofthe bioactive agents. The improved stability involves, for example, themaintenance of a relatively balanced condition, and may be exemplified,for example, by increased resistance of the composition againstdestruction, decomposition, degradation, and the like. In the case ofembodiments involving vesicles filled with gases, gaseous precursorsand/or bioactive agents, the stabilizing compounds may serve to eitherform the vesicles or stabilize the vesicles, in either way serving tominimize or substantially (including completely) prevent the escape ofgases, gaseous precursors and/or bioactive agents from the vesiclesuntil release is desired. The term "substantially," as used in thepresent context of preventing escape of gases, gaseous precursors and/orbioactive agents from the vesicles, means

greater than about 50% is maintained entrapped in the vesicles untilrelease is desired, and preferably greater than about 60%, morepreferably greater than about 70%, even more preferably greater thanabout 80%, still even more preferably greater than about 90%, ismaintained entrapped in the vesicles until release is desired. Inparticularly preferred embodiments, greater than about 95% of the gases,gaseous precursors, and/or bioactive agents are maintained entrappeduntil release is desired. The gases, gaseous precursors and/or bioactiveagents may also be completely maintained entrapped (i.e., about 100% ismaintained entrapped), until release is desired. The resulting mixture,suspension, emulsion or the like may comprise walls (i.e., films,membranes and the like) around the bioactive agent, gases and/or gaseousprecursors, or may be substantially devoid of walls or membranes, ifdesired. The stabilizing may, if desired, form droplets. The stabilizingmaterial may also comprise salts and/or sugars. In other embodiments,the stabilizing materials may be substantially (including completely)crosslinked. The stabilizing material may have a neutral, positive ornegative charge.

"Vesicle stability" refers to the ability of vesicles to retain the gas,gaseous precursor and/or other bioactive agent entrapped therein afterbeing exposed, for about one minute, to a pressure of about 100millimeters (mm) of mercury (Hg). Vesicle stability is measured inpercent (%), this being the fraction of the amount of gas which isoriginally entrapped in the vesicle and which is retained after releaseof the pressure. Vesicle stability also includes "vesicle resilience"which is the ability of a vesicle to return to its original size afterrelease of the pressure.

"Droplef" refers to a spherical or spheroidal entity which may besubstantially liquid or which may comprise liquid and solid, solid andgas, liquid and gas, or liquid, solid and gas. Solid materials within adroplet may be, for example, particles, polymers, lipids, proteins, orsurfactants.

"Covalent association" refers to an intermolecular association or bondwhich involves the sharing of electrons in the bonding orbitals of twoatoms.

"Noncovalent association" refers to intermolecular interaction among twoor more separate molecules which does not involve a covalent bond.Intermolecular interaction is dependent upon a variety of factors,including, for example, the polarity of the involved molecules, and thecharge (neutral, positive or negative) of the involved molecules.Noncovalent associations include, for example, ionic interactions,electrostatic interactions, dipole-dipole interactions, van der Waal'sforces, hydrogen bonding and combinations thereof

"Ionic interaction" or "electrostatic interaction" refers tointermolecular interaction among two or more molecules, each of which ispositively or negatively charged. Thus, for example, "ionic interaction"or "electrostatic interaction" refers to the attraction between a first,positively charged molecule and a second, negatively charged molecule.Ionic or electrostatic interactions include, for example, the attractionbetween a negatively charged bioactive agent, for example, geneticmaterial, and a positively charged lipid, for example, a cationic lipid,such as lauryltrimethylammonium bromide.

"Dipole-dipole interaction" refers generally to the attraction which canoccur among two or more polar molecules. Thus, "dipole-dipoleinteraction" refers to the attraction of the uncharged, partial positiveend of a first polar molecule, commonly designated as δ⁺, to theuncharged, partial negative end of a second polar molecule, commonlydesignated as δ⁻. Dipole-dipole interactions are exemplified by theattraction between the electropositive head group, for example, thecholine head group, of phosphatidylcholine and an electronegative atom,for example, a heteroatom, such as oxygen, nitrogen or sulphur, which ispresent in a stabilizing material, such as a polysaccharide."Dipole-dipole interaction" also refers to intermolecular hydrogenbonding in which a hydrogen atom serves as a bridge betweenelectronegative atoms on separate molecules and in which a hydrogen atomis held to a first molecule by a covalent bond and to a second moleculeby electrostatic forces.

"Van der Waal's forces" refers to the attractive forces betweennon-polar molecules that are accounted for by quantum mechanics. Van derWaal's forces are generally associated with momentary dipole momentswhich are induced by neighboring molecules and which involve changes inelectron distribution.

"Hydrogen bond" refers to an attractive force, or bridge, which mayoccur between a hydrogen atom which is bonded covalently to anelectronegative atom, for example, oxygen, sulfur, or nitrogen, andanother electronegative atom. The hydrogen bond may occur between ahydrogen atom in a first molecule and an electronegative atom in asecond molecule (intermolecular hydrogen bonding). Also, the hydrogenbond may occur between a hydrogen atom and an electronegative atom whichare both contained in a single molecule (intramolecular hydrogenbonding).

"Hydrophilic interaction" refers to molecules or portions of moleculeswhich may substantially bind with, absorb and/or dissolve in water. Thismay result in swelling and/or the formation of reversible gels."Hydrophobic interaction" refers to molecules or portions of moleculeswhich do not substantially bind with, absorb and/or dissolve in water.

"Biocompatible" refers to materials which are generally not injurious tobiological functions and which will not result in any degree ofunacceptable toxicity, including allergenic responses and diseasestates.

"In combination with" refers to the incorporation of bioactive agentsand/or targeting ligands in a stabilizing composition of the presentinvention, including, for example, emulsion, suspensions and vesicles.The bioactive agent and/or targeting ligand can be combined with thestabilizing compositions in any of a variety of ways. For example, thebioactive agent and/or targeting ligand may be associated covalentlyand/or non-covalently with the compounds or stabilizing materials. Inthe case of vesicles, the bioactive agent and/or targeting ligand may beentrapped within the internal void of the vesicle; may be integratedwithin the layer(s) or wall(s) of the vesicle, for example, by beinginterspersed among stabilizing materials which form or are containedwithin the vesicle layer(s) or wall(s); may be located on the surface ofa vesicle or non-vesicular stabilizing material; and/or any combinationthereof. Preferably, the targeting ligand is located on the surface of avesicle or non-vesicular stabilizing material. In any case, thebioactive agent and/or targeting ligand may interact chemically with thewalls of the vesicles, including, for example, the inner and/or outersurfaces of the vesicle and may remain substantially adhered thereto.Such interaction may take the form of, for example, noncovalentassociation or bonding, ionic interactions, electrostatic interactions,dipole--dipole interactions, hydrogen bonding, van der Waal's forces,covalent association or bonding, crosslinking or any other interaction,as will be apparent to one skilled in the art in view of the presentdisclosure. The interaction may result in the stabilization of thevesicle. The bioactive agent and/or targeting ligand may also interactwith the inner or outer surface of the vesicle or the non-vesicularstabilizing material in a limited manner. Such limited interaction wouldpermit migration of the bioactive agent and/or targeting ligand, forexample, from the surface of a first vesicle to the surface of a secondvesicle, or from the surface of a first non-vesicular stabilizingmaterial to a second non-vesicular stabilizing material. Alternatively,such limited interaction may permit migration of the bioactive agentand/or targeting ligand, for example, from within the walls of a vesicleand/or non-vesicular stabilizing material to the surface of a vesicleand/or non-vesicular stabilizing material, and vice versa, or frominside a vesicle or non-vesicular stabilizing material to within thewalls of a vesicle or non-vesicular stabilizing material and vice versa.

"Tissue" refers generally to specialized cells which may perform aparticular function. The term "tissue" may refer to an individual cellor a plurality or aggregate of cells, for example, membranes or organs.The term"tissue" also includes reference to an abnormal cell or aplurality of abnormal cells. Exemplary tissues include pulmonary tissue,myocardial tissue, including myocardial cells and cardiomyocites,membranous tissues, including endothelium and epithelium, laminae,blood, connective tissue, including interstitial tissue, and tumors."Cell" refers to any one of the minute protoplasmic masses which make uporganized tissue, comprising a mass of protoplasm surrounded by amembrane, including nucleated and unnucleated cells and organelles."Receptor" refers to a molecular structure within a cell or on thesurface of a cell which is generally characterized by the selectivebinding of a specific substance. Exemplary receptors includecell-surface receptors for peptide hormones, neurotransmitters,antigens, complement fragments, immunoglobulins and cytoplasmicreceptors.

The present invention describes compositions which comprise one or morecharged lipids, counter ions and at least one lipid which is covalentlybonded to a polymer. An illustrative example of one embodiment of thecompositions of the present invention is presented in FIG. 1A.Preferably, the counter ion in the compositions is divalent in charge orgreater. The compositions may optionally comprise one or more neutrallipids. Additionally, the compositions of the present inventionpreferably comprise a targeting ligand. An illustrative example ofanother embodiment of the compositions of the present inventioncomprising a targeting ligand is presented in FIG. 1B.

In the compositions of the present invention, the charged lipid may bepresent in an amount of from about 10 mole % to about 90 mole %,preferably from about 50 mole % to about 80 mole %, based on the totalamount of lipid in the composition. The counter ion may be present in anamount necessary to balance the charge on the charged lipid.Accordingly, the amount of counter ion in the composition will dependupon the total net charge of the lipid molecule and upon the valency ofthe counter ion. Thus, as one skilled in the art would recognize, forevery equivalent of charges on the charged lipid, about the same numberof equivalents of counter ion should be used. For example, about 1 moleof phosphatidic acid may be used per about 1 mole of Ca²⁺, while about 2moles of stearic acid may be used per about 1 mole of Ca²⁺.

The lipid covalently bonded to polymer may be used in an amount of fromabout 1 mole % to about 50 mole %, preferably from about 1 mole % toabout 25 mole %, more preferably from about 5 mole % to about 25 mole %,based on the total amount of lipid in the composition. If desired,neutral lipids may be used in an amount up to about 10 mole % of thetotal amount of lipid in the composition.

Without intending to be bound by any theory of the invention, in thecompositions of the present invention, the counter ions form saltbridges which crosslink the charged lipids to form aggregates ormultilamellar vesicles. The aggregates or multilamellar vesicles may bereferred to as cochleates, which may be in the form of a tubule or aspiral. The crosslinking of the counter ions may be noncovalent and maygenerally be considered an ionic or electrostatic interaction. The lipidcovalently bonded to the polymer stabilizes the compositions so thatthey from well-defined vesicles. If the lipid covalently bonded to thepolymer is not used in the compositions of the present invention, thecounter ions cause the charged lipid species to form amorphous lipidclumps. In many cases, the lipid clumps may take the form of, forexample, condensed lipid bilayers, but the lipid clumps do not formstable vesicles with size distributions suitable, for example, forintravenous injection.

The lipid covalently bonded to a polymer (e.g., DPPE-PEG-5,000) causescompaction of the size of the composition in the presence of a counterion, such as Ca²⁺ (FIGS. 2B, 3B and 4B), when compared to thecorresponding compositions that do not contain a counter ion (FIGS. 2A,3A and 4A). The compaction effect caused by the lipid covalently bondedto the polymer is most notable when the counter ion is added at theinitial incubation of the lipid mixture. Accordingly, in the methodsdescribed herein for preparing the compositions of the presentinvention, it is preferable to add the counter ion at the initialincubation of the lipid mixture. Increasing the amount of the lipidcovalently bonded to a polymer allows the composition to stabilize atsizes generally under about 1.0 μm in the presence of a counter ion.When the lipid covalently bonded to the polymer is present in an amountless than about 5%, the composition is generally unstable and mayprecipitate. As shown in FIG. 2B, if a counter ion is added afterinitial incubation of the lipid mixture, there does not appear to be anystatistical difference in size of the composition, with or without acounter ion, and with or without the lipid covalently bonded to apolymer.

In the compositions of the present invention, the charged lipid may beanionic (i.e., negatively charged, that is, carrying a net negativecharge) or cationic (i.e., positively charged, that is, carrying a netpositive charge). Preferred anionic lipids include phosphatidic acid,phosphatidyl glycerol and fatty acid esters thereof. Other anioniclipids include amides of phosphatidyl ethanolamine such as anandamidesand methanandamides, phosphatidyl serine, phosphatidyl inositol andfatty acid esters thereof, cardiolipin, phosphatidyl ethylene glycol,acidic lysolipids, palmitic acid, stearic acid, arachidonic acid, oleicacid, linolenic acid, linoleic acid, myristic acid, sulfolipids andsulfatides, free fatty acids, both saturated and unsaturated, andnegatively charged derivatives thereof. More preferably, the anioniclipid is a phosphatidic acid, a phosphatidyl glycerol, a phosphatidylglyercol fatty acid ester, a phosphatidyl ethanolamine anandamide, aphosphatidyl ethanolamine methanandamide, a phosphatidyl serine, aphosphatidyl inositol, a phosphatidyl inositol fatty acid ester, acardiolipin, a phosphatidyl ethylene glycol, an acidic lysolipid, asulfolipid, a sulfatide, a saturated free fatty acid, an unsaturatedfree fatty acid, a palmitic acid, a stearic acid, an arachidonic acid,an oleic acid, a linolenic acid, a linoleic acid or a myristic acid. Ina preferred embodiment, the anionic lipid in the composition of thepresent invention is a fluorinated anionic lipid. Any of the anioniclipids described herein may be fluorinated by replacing at least onehydrogen atom with a fluorine atom. One skilled in the art willrecognize that countless other natural and synthetic variants carryingnegative charged moieties will also function in the invention.

For the anionic lipids, the chain length of the fatty acyl moiety canvary from about 8 to about 26 carbon atoms in length. The lipids may besaturated, monounsaturated or polyunsaturated. Preferably, the lipidchain length ranges from about 10 to about 24 carbon atoms, morepreferably about 16 carbon atoms. A wide variety of anionic lipids maybe used but particularly preferred are dipalmitoylphosphatidic acid(DPPA) and dipalmitoylphosphatidyl glycerol. The carbon chain lengths ofthe various lipids in the compositions of the present invention (e.g.,charged lipid, lipid covalently boned to polymer, neutral lipid) may bethe same or different. Preferably, the chain length of the differentlipids in the composition is similar. In a preferred embodiment, theanionic lipid in the composition of the present invention is afluorinated anionic lipid. Any of the anionic lipids described hereinmay be fluorinated by replacing at least one hydrogen atom with afluorine atom.

When the charged lipid is anionic, a cationic counter ion is used toform the compositions. Suitable cationic counter ions include, forexample, alkaline earths, beryllium (Be⁺²), magnesium (Mg⁺²), calcium(Ca⁺²), strontium (Sr⁺²) and barium (Ba⁺²); amphoteric ions: aluminum(Al⁺³), gallium (Ga⁺³), germanium (Ge⁺³), tin (Sn⁺⁴) and lead (Pb⁺² andPb⁺⁴); transition metals: titanium (Ti⁺³ and Ti⁺⁴), vanadium (V⁺² andV⁺³), chromium (Cr⁺² and Cr⁺³), manganese (Mn⁺² and Mn⁺³), iron (Fe⁺²and Fe⁺³), cobalt (Co⁺² and Co⁺³), nickel (Ni⁺² and Ni⁺³), copper(Cu⁺²), zinc (Zn⁺²), zirconium (Zr⁺⁴), niobium (Nb⁺³), molybdenum (Mo⁺²and Mo⁺³), cadmium (Cd⁺²), indium (In⁺³), tungsten (W⁺² and W⁺⁴), osmium(Os⁺², Os⁺³ and Os⁺⁴), iridium (Ir⁺², Ir⁺³ and Ir⁺⁴), mercury (Hg⁺²) andbismuth (Bi⁺³); and rare earth lanthanides, exemplified by lanthanum(La⁺³) and gadolinium (Gd⁺³). Some of these ions, notably lead andnickel, may be inappropriate for in vivo use due to toxicity. Preferredcations are calcium (Ca⁺²), magnesium (Mg⁺²) and zinc (Zn⁺²) andparamagnetic cations such as manganese (preferably Mn⁺²) and gadolinium(Gd⁺³). Most preferably the cation is calcium (Ca⁺²).

When the charged lipids of the invention carry a net positive charge atpH 7, the lipid is a cationic lipid. Cationic lipids which may be usedin the compositions of the present invention include, for example,phosphatidylethanolamine, phospatidylcholine,glycero-3-ethylphosphatidylcholine and fatty acyl esters thereof, di-and trimethyl ammonium propane, di- and tri-ethylammonium propane andfatty acyl esters thereof. A preferred derivative from this group isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium chloride("DOTMA"). Additionally, a wide array of synthetic cationic lipids canfunction in the present invention. These include common natural lipidsderivatized to contain one or more basic functional groups. Examples oflipids which can be so modified include, for example,dimethyldioctadecylammonium bromide, sphingolipids, sphingo-myelin,lysolipids, glycolipids such as ganglioside GM1, sulfatides,glycosphingolipids, cholesterol and cholesterol esters and salts,N-succinyldioleoyl-phosphatidylethanolamine, 1,2, -dioleoyl-sn-glycerol,1,3-dipalmitoyl-2-succinylglycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine andpalmitoylhomocystiene. In a preferred embodiment, the cationic lipid inthe composition of the present invention is a fluorinated cationiclipid. Any of the cationic lipids described herein may be fluorinated byreplacing at least one hydrogen atom with a fluorine atom

Specially synthesized cationic lipids also function in the presentinvention, including those compounds of formula (I), formula (II) andformula (III), described in U.S. patent application Ser. No. 08/391,938,filed Feb. 21, 1995, the disclosure of which is hereby incorporated byreference herein in its entirety.

The cationic lipid may be a compound of formula (I): ##STR1## where eachof x, y and z is independently an integer from 0 to about 100; each X₁is independently --O--, --S--, --NR₅ --, --C(═X₂)--, --C(═X₂)--N(R₅)--,--N(R₅)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₅X₂)P(═X₂)--X₂ --; each X₂ is independently O or S; each Y₁ isindependently a phosphate residue, N(R₆)_(a) --, S(R₆)_(a) --, P(R₆)_(a)-- or --CO₂ R₆, wherein a is an integer from 1 to 3; each Y₂ isindependently --N(R₆)_(b) --, --S(R₆)_(b) -- or --P(R₆)_(b) --, whereinb is an integer from 0 to 2; each Y₃ is independently a phosphateresidue, N(R₆)_(a) --, S(R₆)_(a) --, P(R₆)_(a) -- or --CO₂ R₆, wherein ais an integer from 1 to 3; each of R₁, R₂, R₃ and R₄ is independentlyalkylene of 1 to about 20 carbons; each R₅ is independently hydrogen oralkyl of 1 to about 10 carbons; and each R₆ is independently --[R₇ --X₃]_(c) --R₈ or --R₉ --[X₄ --R₁₀ ]_(d) --Q, wherein: each of c and d isindependently an integer from 0 to about 100; each Q is independently aphosphate residue, --N(R₁₁)_(q), --S(R₁₁)_(q), --P(R₁₁)_(q) or --CO₂ R₆,wherein q is an integer from 1 to 3; each of X₃ and X₄ is independently--O--, --S--, --NR₅ --, --C(═X₂)--, --C(═X₂)--N(R₅)--,--N(R₅)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₅X₂)P(═X₂)--X₂ --; each R₇ is independently alkylene of 1 to about 20carbons; each R₈ is independently hydrogen or alkyl of 1 to about 60carbons; each of R₉ and R₁₀ is independently alkylene of 1 to about 20carbons; and each R₁₁ is independently -[R₇ --X₃ ]_(c) --R₈ or --R₉--[X₄ --R₁₀ ]_(d) --W, wherein: each W is independently a phosphateresidue, --N(R₁₂)_(w), --S(R₁₂)_(w), --P(R₁₂)_(w) or --CO₂ R₆, wherein wis an integer from 1 to 3; and R₁₂ is --[R₇ --X₃ ]_(c) --R₈, with theproviso that the compound of formula (I) comprises at least one, andpreferably at least two, quatemary salts.

In the above formula (I), each of x, y and z is independently an integerfrom 0 to about 100. Preferably, each of x, y and z is independently aninteger of from 0 to about 50, with integers from 0 to about 20 beingmore preferred. Even more preferably, each of x, y and z isindependently an integer from 0 to about 10, with integers from 0 toabout 5 being still more preferred. In certain particularly preferredembodiments, x is 1, y is 2or 3 and z is 0 or 1.

In the above formula (I), each X₁ is independently --O--, --S--, --NR₅--, --C(═X₂)--, --C(═X₂)--N(R₅)--, --N(R₅)--C(═X₂)--, --C(═X₂)--O--,--O--C(═X₂)-- or --X₂ --(R₅ X₂)P(═X₂)--X₂ --. Preferably, each X₁ isindependently --C(═O)--NR₅ --, --NR₅ --C(═O)--, --C(═O)--O-- or--O--C(═O)--.

Each X₂ in the definitions of X₁, X₃ and X₄ above is independently O orS. Preferably, X₂ is O.

In the above formula (I), each Y₁ is independently a phosphate residue,N(R₆)_(a) --, S(R₆)_(a) --, P(R₆)_(a) -- or --CO₂ R₆, wherein a is aninteger from 1 to 3. Preferably, each Y₁ is independently a phosphateresidue, N(R₆)_(a) -- or --CO₂ R₆, wherein a is 2 or 3. Preferably, a is3.

Each Y₂ in formula (I) above is independently-N(R₆)_(b) --, --S(R₆)_(b)-- or --P(R₆)_(b) --, wherein b is an integer from 0 to 2. Preferably,Y₂ is --N(R₆)_(b) --, wherein b is 1 or 2.

In the above formula (I), each Y₃ is independently a phosphate residue,N(R₆)_(a) --, S(R₆)_(a) --, P(R₆)_(a) -- or --CO₂ R₆, wherein a is aninteger from 1 to 3. Preferably, each Y₃ is independently a phosphateresidue, N(R₆)_(a) -- or --CO₂ R₆, where a is 2 or 3, preferably a is 3.

In the above formula (I), each of R₁, R₂, R₃ and R₄ is independentlyalkylene of 1 to about 20 carbons. Preferably, each of R₁, R₂, R₃ and R₄is independently straight chain alkylene of 1 to about 10 carbons orcycloalkylene of about 4 to about 10 carbons. More preferably, each ofR₁, R₂, R₃ and R₄ is independently straight chain alkylene of 1 to about4 carbons or cycloalkylene of about 5 to about 7 carbons. Even morepreferably, each of R₁, R₂, R₃ and R₄ is independently methylene,ethylene or cyclohexylene.

In the above definitions of X₁, X₃ and X₄, each R₅ is independentlyhydrogen or alkyl of 1 to about 10 carbons. Preferably, each R₅ isindependently hydrogen or alkyl of 1 to about 4 carbons. Morepreferably, R₅ is hydrogen.

In the above definitions of Y₁, Y₂ and Y₃, each R₆ is independently--[R₇ --X₃ ]_(c) --R₈ or --R₉ --[X₄ --R₁₀ ]_(d) --Q, wherein each of cand d is independently an integer from 0 to about 100. Preferably, eachof c and d is independently an integer from 0 to about 50, with integersfrom 0 to about 20 being more preferred. Even more preferably, each of cand d is independently an integer from 0 to about 10, with integers from0 to about 5 being still more preferred. In certain particularlypreferred embodiments, c is 0 or 1 and d is 1.

Each Q in R₆ above is independently a phosphate residue, --N(R₁₁)_(q),--S(R₁₁)_(q), --P(R₁₁)_(q) or --CO₂ R₁₁, wherein q is an integer from 1to 3. Preferably, each Q is independently a phosphate residue,--N(R₁₁)_(q) or --CO₂ R₁₁, where q is 2 or 3 preferably 3.

Also in the above definition of R₆, each of X₃ and X₄ is independently--O--, --S--, --NR₅ --, --C(═X₂)--, --C(═X₂)--N(R₅)--,--N(R₅)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₅X₂)P(═X₂)--X₂ --, wherein each of X₂ and R₅ is independently aspreviously described. Preferably, each of X₃ and X₄ is independently--C(═O)--NR₅ --, --NR₅ --C(═O)--, --C(═O)--O-- or --O--C(═O)--.

In the definitions of R₆, R₁₁ and R₁₂ above, each R₇ is independentlyalkylene of 1 to about 20 carbons. Preferably, each R₇ is independentlyalkylene of 1 to about 10 carbons, with alkylene of 1 to about 4 carbonsbeing preferred. More preferably, each R₇ is independently methylene orethylene.

Also in the definitions of R₆, R₁₁ and R₁₂ above, each R₈ isindependently hydrogen or alkyl of 1 to about 60 carbons. Preferably,each R₈ is independently hydrogen or alkyl of 1 to about 40 carbons,with hydrogen or alkyl of 1 to about 20 carbons being more preferred.Even more preferred, each R₈ is independently hydrogen or alkyl of 1 toabout 16 carbons. In certain particularly preferred embodiments, each R₈is independently hydrogen, methyl, dodecyl or hexadecyl.

Each of R₉ and R₁₀ in the definitions of R₆ and R₁₁ above isindependently alkylene of 1 to about 20 carbons. Preferably, each of R₉and R₁₀ is independently alkylene of 1 to about 10 carbons. Morepreferably, each of R₉ and R₁₀ is independently alkylene of 1 to about 4carbons. Even more preferably, each of R₉ and R₁₀ is independentlymethylene or ethylene.

Each R₁₁ in Q above is independently --[R₇ --X₃ ]_(c) --R₈ or --R₉ --[X₄--R₁₀ ]_(d) --W, wherein each of c, d, X₃, X₄, R₇, R₈, R₉ and R₁₀ isindependently as previously described.

Each W in R₁₁ above is independently a phosphate residue, --N(R₁₂)_(w),--S(R₁₂)_(w), --P(R₁₂)_(w) or --CO₂ R₁₂, wherein w is an integer from 1to 3. Preferably, W is a phosphate residue, --N(R₁₂)_(w) or --CO₂ R₁₂,wherein w is 2 or 3. Preferably, w is 3.

In the above definition of W, R₁₂ is --[R₇ --X₃ ]_(c) --R₈, wherein eachof c, X₃, R₇ and R₈ is independently as previously described.

Another cationic lipid compound is a compound of the formula (II):

    Y.sub.1 --R.sub.1 --Y.sub.1                                (II)

where each Y₁ is independently a phosphate residue, N(R₂)_(a) --,S(R₂)_(a) --, P(R₂)_(a) -- or --CO₂ R₂, wherein a is an integer from 1to 3; R₁ is alkylene of 1 to about 60 carbons containing 0 to about 30--O--, --S--, --NR₃ -- or --X₂ --(R₃ X₂)P(═X₂)--X₂ -- heteroatoms orheteroatom groups; R₂ a residue of the formula --R₄ --[(X₁ --R₅)_(x)--Y₂ ]_(y) --R₆, wherein each of x and y is independently an integerfrom 0 to about 100; each X₁ is independently a direct bond, --O--,--S--, --NR₃ --, --C(═X₂)--, --C(═X₂)--N(R₃)--, --N(R₃)--C(═X₂)--,--C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₃ X₂)P(═X₂)--X₂ --; each X₂ isindependently O or S; each Y₂ is independently --S(R₂)_(b) --,--N(R₂)_(b) -- or --P(R₂)_(b) --, wherein b is an integer from 0 to 2;each R₃ is independently hydrogen or alkyl of 1 to about 10 carbons;each of R₄ and R₅ is independently a direct bond or alkylene of 1 toabout 30 carbons containing 0 to about 15 --O--, --S--, --NR₃ -- or --X₂--(R₃ X₂)P(═X₂)--X₂ -- heteroatoms or heteroatom groups; and each R₆ isindependently hydrogen or alkyl of 1 to about 60 carbons containing 0 toabout 30 --O--, --S--, --NR₃ -- or --X₂ --(R₃ X₂)P(═X₂)--X₂ --heteroatoms or heteroatom groups; with the proviso that the compound offormula (II) comprises at least one, and preferably at least two,quaternary salts.

In the above formula (II), each Y₁ is independently a phosphate residue,N(R₂)_(a) --, S(R₂)_(a) --, P(R₂)_(a) -- or --CO₂ R₂, wherein a is aninteger from 1 to 3. Preferably, each Y₁ is independently a phosphateresidue, --N(R₂)_(a) -- or --CO₂ R₂, wherein a is 2 or 3. Preferably, ais 3.

Also in the above formula (II), R₁ is alkylene of 1 to about 60 carbonscontaining 0 to about 30 --O--, --S--, --NR₃ -- or --X₂ --(R₃X₂)P(═X₂)--X₂ -- heteroatoms or heteroatom groups. Preferably, R₁ isalkylene of 1 to about 40 carbons, with alkylene of 1 to about 20carbons being preferred. More preferably, R₁ is straight chain alkyleneof 1 to about 10 carbons or cycloalkylene of about 4 to about 10carbons. Even more preferably, R₁ is straight chain alkylene of 1 toabout 4 carbons or cycloalkylene of about 5 to about 7 carbons.

In the above definition of Y₁, R₂ is a residue of the formula --R₄--[(X₁ --R₅)_(x) --Y₂ ]_(y) --R₆, wherein each of x and y isindependently an integer from 0 to about 100. Preferably, each of x andy is independently an integer from 0 to about 50, with integers from 0to about 20 being more preferred. Even more preferably, each of x and yis independently an integer from 0 to about 10.

In the above definition of R₂, each X₁ is independently a direct bond,--O--, --S--, --NR₃ --, --C(═X₂)--, --C(═X₂)--N(R₃)--,--N(R₃)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₃X₂)P(═X₂)--X₂ --. Preferably, X₁ is a direct bond, --C(═X₂)--N(R₃)--,--N(R₃)--C(═X₂)--, --C(═X₂)--O-- or --O--C(═X₂)--.

Each X₂ in the above definitions of X₁, R₁, R₄, R₅ and R₆ isindependently O or S. Preferably, X₂ is O.

Each Y₂ in the above definition of R₂ is independently --S(R₂)_(b) --,--N(R₂)_(b) -- or --P(R₂)_(b) --, wherein b is an integer of from 0 to2. Preferably, Y₂ is --N(R₂)_(b) -- and b is 1 or 2.

In the above definitions of X₁, R₁, R₄, R₅ and R₆, each R₃ isindependently hydrogen or alkyl of 1 to about 10 carbons. Preferably,each R₃ is independently hydrogen or alkyl of 1 to about 4 carbons. Morepreferably, R₃ is hydrogen.

In the above definition of R₂, each of R₄ and R₅ is independently adirect bond or alkylene of 1 to about 30 carbons containing 0 to about15 --O--, --S--, --NR₃ -- or --X₂ --(R₃ X₂)P(═X₂)--X₂ -- heteroatoms orheteroatom groups. Preferably, each of R₄ and R₅ is independently adirect bond or alkylene of 1 to about 20 carbons. More preferably, eachof R₄ and R₅ is independently a direct bond, straight chain alkylene of1 to about 10 carbons or cycloalkylene of 4 to about 10 carbons. Evenmore preferably, each of R₄ and R₅ is independently a direct bond,straight chain aikylene of 1 to about 4 carbons or cycloalkylene ofabout 5 to about 7 carbons.

Each R₆ in R₂ above is independently hydrogen or alkyl of 1 to about 60carbons containing 0 to about 30 --O--, --S--, --NR₃ -- or --X₂ --(R₃X₂)P(═X₂)--X₂ -- heteroatoms or heteroatom groups. Preferably, each R₆is independently hydrogen or alkyl of 1 to about 40 carbons. Morepreferably, each R₆ is independently hydrogen or alkyl of 1 to about 20carbons.

The cationic lipid may also be a compound of the formula (III): ##STR2##where each of x, y and z is independently an integer from 0 to about100; each X₁ is independently --O--, --S--, --NR₅ --, --C(═X₂)--,--C(═X₂)--N(R₅)--, --N(R₅)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or--X₂ --(R₅ X₂)P(═X₂)--X₂ --; each X₂ is independently O or S; each Y₁independently --O--, --N(R₆)_(a) --, --S(R₆)_(a) -- or --P(R₆)_(a) --,wherein a is an integer from 0 to 2; each Y₂ is independently--N(R₆)_(a) --, --S(R₆)_(a) -- or --P(R₆)_(a) --, wherein a is aninteger from 0 to 2; each Y₃ is independently a phosphate residue,N(R₆)_(b) --, S(R₆)_(b) --, P(R₆)_(b) -- or --CO₂ R₆, wherein b is aninteger from 1 to 3; each of R₁, R₂, R₃ and R₄ is independently alkyleneof 1 to about 20 carbons; each R₅ is independently hydrogen or alkyl of1 to about 10 carbons; and each R₆ is independently --[R₇ --X₃ ]_(c)--R₈ or --R₉ --[X₄ --R₁₀ ]_(d) --Q, where each of c and d isindependently an integer from 0 to about 100; each Q is independently aphosphate residue, --N(R₁₁)_(q), --S(R₁₁)_(q), --P(R₁₁)_(q) or --CO₂R₁₁, wherein q is an integer from 1 to 3; each of X₃ and X₄ isindependently --O--, --S--, --NR₅ --, --C(═X₂)--, --C(═X₂)--N(R₅)--,--N(R₅)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₅X₂)P(═X₂)--X₂ --; each R₇ is independently alkylene of 1 to about 20carbons; each R₈ is independently hydrogen or alkyl of 1 to about 60carbons; each of R₉ and R₁₀ is independently alkylene of 1 to about 20carbons; and each R₁₁ is independently --[R₇ --X₃ ]_(c) --R₈ or --R₉--[X₄ --R₁₀ ]_(d) --W, where each W is independently a phosphateresidue, --N(R₁₂)_(w), --S(R₁₂)_(w), --P(R₁₂)_(w) or --CO₂ R₁₂, whereinw is an integer from 1 to 3; and R₁₂ is --[R₇ --X₃ ]_(c) --R₈ ; with theproviso that the compound of formula (III) comprises at least one, andpreferably at least two, quaternary salts.

In the above formula (III), each of x, y and z is independently aninteger from 0 to about 100. Preferably, each of x, y and z isindependently an integer from 0 to about 50, with integers from 0 toabout 20 being more preferred. Even more preferably, each of x, y and zis independently an integer from 0 to about 10. Still more preferably,each of x, y and z is independently an integer from 0 to about 5. Incertain particularly preferred embodiments, x is 1, y is 2 or 3 and z is0 or 1.

In the above formula (III), each X₁ is independently --O--, --S--, --NR₅--, --C(═X₂)--, --C(═X₂)--N(R₅)--, --N(R₅)--C(═X₂)--, --C(═X₂)--O--,--O--C(═X₂)-- or --X₂ --(R₅ X₂)P(═X₂)--X₂ --. Preferably, each X₁ isindependently --C(═O)--NR₅ --, --NR₅ --C(═O)--, --C(═O)--O-- or--O--C(═O)--.

In the above definitions of X₁, X₃ and X₄, each X₂ is independently O orS. Preferably, X₂ is O.

Each Y₁ in formula (III) above is independently --O--, --N(R₆)_(a) --,--S(R₆)_(a) -- or --P(R₆)_(a) --, wherein a is an integer from 0 to 2.Preferably, Y₁ is --N(R₆)_(a) --, where a is 1 or 2.

Each Y₂ in formula (III) above is independently --N(R₆)_(a) --,--S(R₆)_(a) -- or --P(R₆)_(a) --, wherein a is an integer from 0 to 2.Preferably, Y₂ is --N(R₆)_(a) --.

In the above formula (III), each Y₃ is independently a phosphateresidue, N(R₆)_(b) --, S(R₆)_(b) --, P(R₆)_(b) -- or --CO₂ R₆, wherein bis an integer from 1 to 3. Preferably, each Y₃ is independently aphosphate residue or N(R₆)_(b) --, wherein b is 2 or 3. Preferably, b is3.

In the above formula (III), each of R₁, R₂, R₃ and R₄ is independentlyalkylene of 1 to about 20 carbons. Preferably, each of R₁, R₂, R₃ and R₄is independently straight chain alkylene of 1 to about 10 carbons orcycloalkylene of about 4 to about 10 carbons. More preferably, each ofR₁, R₂, R₃ and R₄ is independently straight chain alkylene of 1 to about4 carbons or cycloalkylene of about 5 to about 7 carbons. Even morepreferably, each of R₁, R₂, R₃ and R₄ is independently methylene,ethylene or cyclohexylene.

In the above definitions of X₁, X₃ and X₄, each R₅ is independentlyhydrogen or alkyl of 1 to about 10 carbons. Preferably, each R₅ isindependently hydrogen or alkyl of 1 to about 4 carbons. Morepreferably, R₅ is hydrogen.

In the above definitions of Y₁, Y₂ and Y₃, each R₆ is independently--[R₇ --X₃ ]_(c) --R₈ or --R₉ --[X₄ --R₁₀ ]_(d) --Q, wherein each of cand d is independently an integer from 0 to about 100. Preferably, eachof c and d is independently an integer from 0 to about 50, with integersfrom 0 to about 20 being more preferred. Even more preferably, each of cand d is independently an integer from 0 to about 10, with integers from0 to about 5 being still more preferred. In certain particularlypreferred embodiments, c is 0 or 1 and d is 1.

Each Q in R₆ above is independently a phosphate residue, --N(R₁₁)_(q),--S(R₁₁)_(q), --P(R₁₁)_(q) or --CO₂ R₁₁, wherein q is an integer from 1to 3. Preferably, each Q is independently a phosphate residue,--N(R₁₁)_(q) or --CO₂ R₁₁, wherein q is 2 or 3, preferably 3.

Also in the above definition of R₆, each of X₃ and X₄ is independently--O--, --S--, --NR₅ --, --C(═X₂)--, --C(═X₂)--N(R₅)--,--N(R₅)--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)-- or --X₂ --(R₅X₂)P(═X₂)--X₂ --, wherein X₂ and R₅ are as previously described.Preferably, each of X₃ and X₄ is independently --C(═O)--NR₅ --, --NR₅--C(═O)--, --C(═O)--O-- or --O--C(═O)--.

In the definitions of R₆, R₁₁, and R₁₂ above, each R₇ is independentlyalkylene of 1 to about 20 carbons. Preferably, each R₇ is independentlyalkylene of 1 to about 10 carbons, with alkylene of 1 to about 4 carbonsbeing preferred. More preferably, each R₇ is independently methylene orethylene.

Also in the definitions of R₆, R₁₁ and R₁₂ above, each R₈ isindependently hydrogen or alkyl of 1 to about 60 carbons. Preferably,each R₈ is independently hydrogen or alkyl of 1 to about 40 carbons,with hydrogen or alkyl of 1 to about 20 carbons being more preferred. Incertain particularly preferred embodiments, each R₈ is independentlyhydrogen, methyl, dodecyl or hexadecyl.

Each of R₉ and R₁₀ in the definitions of R₆ and R₁₁ above isindependently alkylene of 1 to about 20 carbons. Preferably, each of R₉and R₁₀ is independently alkylene of 1 to about 10 carbons. Morepreferably, each of R₉ and R₁₀ is independently alkylene of 1 to about 4carbons. Even more preferably, each of R₉ and R₁₀ is independentlymethylene or ethylene.

In Q above, each R₁₁ is independently --[R₇ --X₃ ]_(c) --R₈ or --R₉--[X₄ --R₁₀ ]_(d) --W, wherein each of c, d, X₃, X₄, R₇, R₈, R₉ and R₁₀is independently as previously described.

Each W in R₁₁ above is independently a phosphate residue, --N(R₁₂)_(w),--S(R₁₂)_(w), --P(R₁₂)_(w) or --CO₂ R₁₂, wherein w is an integer from 1to 3. Preferably, each W is independently a phosphate residue,--N(R₁₂)_(w) or --CO₂ R₁₂, wherein w is 2 or 3. Preferably, w is 3.

In W above, R₁₂ is --[R₇ --X₃ ]_(c) --R₈, wherein each of c, X₃, R₇ andR₈ is independently as previously described.

In the above formulas (I), (II) and (III), it is intended that when anysymbol appears more than once in a particular formula or substituent,its meaning in each instance is independent of the other. Also in theabove formulas (I), (II) and (III), it is intended that when each of twoor more adjacent symbols is defined as being "a direct bond" to providemultiple, adjacent direct bonds, the multiple and adjacent direct bondsdevolve into a single direct bond.

Specific examples of the above cationic lipids include, for example,N,N'-Bis (dodecyaminocarbonylmethylene)-N,N'-bis(β-N,N,N-trimethylammoniumethyl-aminocarbonylmethylene)-ethylenediaminetetraiodide; N,N"-Bis (hexadecylamino-carbonylmethylene)-N,N',N"-tris(β-N,N,N-trimethylammoniumethylaminocarbonyl-methylenediethylenetriaminehexaiodide; N,N'-Bis(dodecylaminocarbonylmethylene)-N,N"-bis(β-N,N,N-trimethylammoniumethylaminocarbonylmethylene)cyclohexylene-1,4-diaminetetraiodide;1,1,7,7-tetra-(β-N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3-hexadecylaminocarbonylmethylene-1,3,7-triaazaheptaneheptaiodide; and N,N,N'N'-tetra(β-N,N,N-trimethylammoniumethylaminocarbonylmethylene)-N'-(1,2-dioleoylglycero-3-phosphoethanolaminocarbonylmethylene)-diethylenetriaminetetraiodide. In a preferred embodiment, the cationic lipid in thecomposition of the present invention is a fluorinated cationic lipid.Any of the cationic lipids described herein may be fluorinated byreplacing at least one hydrogen atom with a fluorine atom. One skilledin the art will recognize that countless other natural and syntheticvariants carrying positive charged moieties will also function in theinvention.

Anionic counter ions which be used with the cationic lipids include, forexample, ethylene diamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA), and 1, 4, 7,10-tetraazocyclododecane-N', N', N", N"-tetraacetic acid (DOTA). Othernegatively charged species which function as counter ions include, forexample, monatomic and polyatomic anions such as dicarboxylic acids,teraphthalic acid, sulfide ions, sulfite ions, sulfate ions, oxide ions,nitride ions, carbonate ions and phosphate ions; polymers and copolymersof acrylic acid, methacrylic acid, other derivatives of acrylic acid,polymers with pendant SO₃ H groups such as sulfonated polystyrene andpolystyrene with carboxylic acid groups. Preferably, the negativelycharged counter ions are divalent, trivalent or multivalent.

In the compositions of the present invention, the lipids covalentlybonded to polymers include, for example, lipids covalently bonded tohydrophilic polymers. Suitable hydrophilic polymers for covalent bondingto lipids include, for example, polyalkyleneoxides such as, for example,polyethylene glycol (PEG) and polypropylene glycol (PPG),polyvinyl-pyrrolidones, polyvinylalkylethers, such as a polyvinylmethylether, polyacrylamides, such as, for example, polymethacrylamides,polydimethylacrylamides and polyhydroxy-propylmethacrylamides,polyhydroxyalkyl(meth)acrylates, such as polyhydroxyethyl acrylates,polyhydroxypropyl methacrylates, polyalkyloxazolines, such aspolymethyloxazolines and polyethyloxazolines,polyhydroxyalkyloxazolines, such as polyhydroxyethyloxazolines,polyhyhydroxypropyloxazolines, polyvinyl alcohols, polyphosphazenes,poly(hydroxy-alkylcarboxylic acids), polyoxazolidines, polyaspartamide,and polymers of sialic acid (polysialics). Preferably, the hydrophilicpolymers are polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polypropylene glycol, a polyvinylalkylether, a polyacrylamide,a polyalkyloxazoline, a polyhydroxyalkyloxazoline, a polyphosphazene, apolyoxazolidine, a polyaspartamide, a polymer of sialic acid, apolyhydroxyalkyl(meth)acrylate or a poly(hydroxyalkylcarboyxlic acid).More preferably, the hydrophilic polymers are PEG, PPG,polyvinylalcohol, polyvinylpyrrolidone and copolymers thereof, with PEGand PPG polymers being more preferred and PEG polymers being even moreprefered. The polyethylene glycol may be, for example, PEG 2000, PEG5000 or PEG 8000, which have weight average molecular weights of 2000,5000 and 8000 daltons, respectively. Preferably, the polyethylene glycolhas a molecular weight of about 500 to about 20,000, more preferablyfrom about 1,000 to about 10,000. Other suitable polymers, hydrophilicand otherwise, will be apparent to one skilled in the art based on thepresent disclosure. Polymers which may be attached to the lipid viaalkylation or acylation reactions onto the surface of the liposome areparticularly useful for improving the stability and size of thedistribution of the liposomes. Exemplary lipids which are covalentlybonded to hydrophilic polymers include, for example,dipalmitoylphosphatidylethanolamine-PEG,dioleoylphosphatidylethanolamine-PEG anddistearylphosphatidylethanolamine-PEG, more preferablydipalmitoylphosphatidylethanolamine-PEG.

In addition to the anionic and cationic lipids described above, othersuitable lipids which may be used in the present invention include, forexample, fatty acids, lysolipids, fluorinated lipids, phosphocholines,such as those associated with platelet activation factors (PAF) (AvantiPolar Lipids, Alabaster, AL), including 1-alkyl-2-acetoyl-sn-glycero3-phosphocholines, and 1-alkyl-2-hydroxy-sn-glycero 3-phosphocholines,which target blood clots; phosphatidylcholine with both saturated andunsaturated lipids, including dioleoylphosphatidylcholine;dimyristoylphosphatidylcholine (DMPC);dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine;dipalmitoylphosphatidylcholine (DPPC); distearoylphosphatidylcholine(DSPC); and diarachidonylphosphatidylcholine (DAPC);phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine,dimyristoylphosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE) anddistearoylphosphatidylethanolamine (DSPE); phosphatidylserine;phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG);phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipidssuch as ganglioside GM1 and GM2; glucolipids; sulfatides;glycosphingolipids; phosphatidic acids, such as dipalmitoylphosphatidicacid (DPPA) and distearoylphosphatidic acid (DSPA); palmitic acid;stearic acid; arachidonic acid; oleic acid; linolenic acid; linoleicacid; myristic acid; synthetic lipids described in U.S. Pat. No.5,312,617, the disclosure of which is hereby incorporated by referenceherein in its entirety; lipids bearing polymers, such as-chitin,hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), alsoreferred to herein as "pegylated lipids" with preferred lipid bearingpolymers including DPPE-PEG (DPPE-PEG), which refers to the lipid DPPEhaving a PEG polymer attached thereto, including, for example,DPPE-PEG5000, which refers to DPPE having attached thereto a PEG polymerhaving a mean average molecular weight of about 5000; lipids bearingsulfonated mono-, di-, oligo- or polysaccharides; cholesterol,cholesterol sulfate and cholesterol hemisuccinate; tocopherolhemisuccinate; lipids with ether and ester-linked fatty acids;polymerized lipids (a wide variety of which are known in the art);diacetyl phosphate; dicetyl phosphate; stearylamine; cardiolipin;phospholipids with short chain fatty acids of about 6 to about 8 carbonsin length; synthetic phospholipids with asymmetric acyl chains, such as,for example, one acyl chain of about 6 carbons and another acyl chain ofabout 12 carbons; ceramides; non-ionic liposomes including niosomes suchas polyoxyalkylene (e.g., polyoxyethylene) fatty acid esters,polyoxyalkylene (e.g., polyoxyethylene) fatty alcohols, polyoxyalkylene(e.g., polyoxyethylene) fatty alcohol ethers, polyoxyalkylene (e.g.,polyoxyethylene) sorbitan fatty acid esters (such as the class ofcompounds referred to as TWEEN®, including, for example, TWEEN® 20,TWEEN® 40 and TWEEN® 80, commercially available from ICI Americas, Inc.,Wilmington, Del.), glycerol polyethylene glycol oxystearate, glycerolpolyethylene glycol ricinoleate, alkyloxylated (e.g., ethoxylated)soybean sterols, alkyloxylated (e.g., ethoxylated) castor oil,polyoxyethylene-polyoxypropylene polymers, and polyoxyalkylene (e.g.,polyoxyethylene) fatty acid stearates; sterol aliphatic acid estersincluding cholesterol sulfate, cholesterol butyrate, cholesterolisobutyrate, cholesterol palmitate, cholesterol stearate, lanosterolacetate, ergosterol palmitate, and phytosterol n-butyrate; sterol estersof sugar acids including cholesterol glucuronide, lanosterolglucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide,cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate;esters of sugar acids and alcohols including lauryl glucuronide,stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoylgluconate, and stearoyl gluconate; esters of sugars and aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid and polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, anddigitoxigenin; glycerol dilaurate, glycerol trilaurate, glyceroldipalmitate, glycerol and glycerol esters including, glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate; long chain alcohols includingn-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, andn-octadecyl alcohol;6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)-hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside;12-(((7'-diethylaminocoumarin-3-yl)-carbonyl)methylamino)-octadecanoicacid;N-[12-(((7'-diethylaminocoumarin-3-yl)-carbonyl)methylamino)-octadecanoyl]-2-aminopalmiticacid; cholesteryl(4'-trimethylammonio)butanoate;N-succinyldioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycero-phosphoethanolamine andpalmitoylhomocysteine, and/or any combinations thereof. One skilled inthe art could readily determine the charge (e.g., cationic, anionic orneutral) of any of the lipids decribed herein. In a preferredembodiment, the lipids described herein are fluorinated lipids. As oneskilled in the art will recognize, any of the neutral lipids describedherein may be modified to cationic lipids or anionic lipids by methodsthat are well-known to one skilled in the art. For example, anymodifiable group on a neutral lipid, such as a secondary amine, an --OHgroup or an anionic group or cationic group that have a zwitterioniccharge balance, may be chemically modified to add or subtract a chargeto the neutral lipid. When a neutral lipid is used in the compositionsof the present invention, the neutral lipid is preferably aphosphocholine, a sphingolipid, a glycolipid, a glycosphingolipid, aphospholipid or a polymerized lipid.

Examples of polymerized lipids include unsaturated lipophilic chainssuch as alkenyl or alkynyl, containing up to about 50 carbon atoms.Further examples are phospholipids such as phosphoglycerides andsphingolipids carrying polymerizable groups; and saturated andunsaturated fatty acid derivatives with hydroxyl groups, such as forexample triglycerides of d-1 2-hydroxyoleic acid, including castor oiland ergot oil. Polymerization may be designed to include hydrophilicsubstituents such as carboxyl or hydroxyl groups, to enhancedispersability so that the backbone residue resulting frombiodegradation is water soluble. Suitable polymerizable lipids are alsodescribed, for example, by Klaveness et al, U.S. Pat. No. 5,536,490, thedisclosure of which is hereby incorporated by reference herein in itsentirety.

Suitable fluorinated lipids that my be used in the compositions of thepresent include, for example, compounds of the formula:

    C.sub.n F.sub.2n+1 (CH.sub.2).sub.m C(O)OOP(OO.sup.-)O(CH.sub.2).sub.w N.sup.+ (CH.sub.3).sub.3 C.sub.n F.sub.2n+1 (CH.sub.2).sub.m C(O)O

where m is 0 to about 18, n is 1 to about 12; and w is 1 to about 8.Examples of and methods for the synthesis of these, as well as otherfluorinated lipids useful in the present invention, are set forth inU.S. application Ser. No. 08/465,868, filed Jun. 6, 1995, Reiss et al,U.S. Pat. No. 5,344,930, Frezard et al, Biochem Biophys Acta, 1192:61-70(1994), and Frezard et al, Art. Cells Blood Subs and Immob Biotech.,22:1403-1408 (1994), the disclosures of each of which are incorporatedherein by reference in their entirety. One specific example of adifluoroacyl glycerylphosphatidylcholine, nonafluorinated diacylglycerylphosphatidylcholine, is represented by compound A, below. Oneskilled in the art will appreciate that analogous fluorinatedderivatives of other common phospholipids (diacylphosphatidyl serine,diacylphosphatidyl ethanolamine, diacylphosphatidyl glycerol,diacylphosphatidyl glycerol, and the like) as well as fluorinatedderivatives of fatty acyl esters and free fatty acids may also functionin accordance with the scope of the invention. Additionally lipid basedand fluorinated (including perfluorinated) surfactants may be used asstabilizing materials in the present invention.

Exemplary polymerizable and/or fluorinated lipid compounds which may beused in the compositions of the present invention are presented below.##STR3## In formula A, above, x is an integer from about 8 to about 18,and n is 2x. Most preferably x is 12 and n is 24. In formulas B, C, Kand L, above, m, n, m' and n' are, independently, an integer of fromabout 8 to about 18, preferably about 10 to about 14.

Other lipids which may be used in the present invention includefluorinated (including perfluorinated) lipid compounds. Suitablefluorinated lipid compounds include, for example, fluorinatedsurfactants, including alkyl surfactants, and amphiphilic compounds. Awide variety of such compounds may be employed, including, for example,the class of compounds which are commercially available as ZONYL®fluorosurfactants (the DuPont Company, Wilmington, Del.), including theZONYL® phosphate salts (e.g., [F(CF₂ CF₂)₃₋₈ CH₂ CH₂ O]₁,2 P(O)(O⁻ NH₄⁺)₂,1) which have terminal phosphate groups and ZONYL® sulfate saltswhich have terminal sulfate groups (e.g., F(CF₂ CF₂)₃₋₈ CH₂ CH₂ SCH₂ CH₂N⁺ (CH₃)₃ ⁻ OSO₂ OCH₃). Suitable ZONYL® surfactants also include, forexample, ZONYL® fluorosurfactants identified as Telomer B, includingTelomer B fluorosurfactants which are pegylated (i.e., have at least onepolyethylene glycol group attached thereto), also known as PEG-TelomerB, available from the DuPont Company. Other suitable fluorosurfactantsare described in U.S. Pat. Nos. 5,276,146, 5,344,930, and 5,562,893, andU.S. application Ser. No. 08/465,868, filed Jun. 6, 1995, thedisclosures of each of which are hereby incorporated by reference hereinin their entirety.

It may also be desirable to use a fluorinated liquid compound,especially a perfluorocarbon compound or a perfluoroether compound,which is in the liquid state at the temperature of use, including, forexample, the in vivo temperature of the human body, to assist or enhancethe stability of the lipid and/or vesicle compositions, and especially,gas filled vesicles. Suitable liquid perfluorocarbons and liquidperfluoroethers include, for example, perfluorohexane,perfluorocyclohexane, perfluoroheptane, perfluorooctane,perfluorononane, perfluorodecane, perfluorodecalin, perfluorododecalin,perfluorooctyliodide, perfluorooctylbromide, perfluorotripropylamine,perfluorotributylamine, perfluorobutylethyl ether,bis(perfluoroisopropyl) ether, and bis(perfluoropropyl) ether. Ingeneral, perfluorocarbons and perfluoroethers comprising about six ormore carbon atoms will be liquids at normal human body temperature.Among these, perfluorooctylbromide and perfluorohexane, which areliquids at room temperature, are preferred. Although not intending to bebound by any theory of operation, in the case of vesicle compositions,the liquid fluorinated compound may be situated at the interface betweenthe gas and the membrane or wall surface of the vesicle. Thus, anadditional stabilizing layer of liquid fluorinated compound may beformed on the internal surface of the stabilizing composition, and thisperfluorocarbon layer may also prevent the gas from diffusing throughthe vesicle membrane. Preferred perfluorinated surfactants are partiallyfluorinated phosphocholine surfactants. In these preferred fluorinatedsurfactants, the dual alkyl compounds may be fluorinated at the terminalalkyl chains and the proximal carbons may be hydrogenated. Thesefluorinated phosphocholine surfactants may be used for making thestabilizing materials and/or vesicles of the present invention.

Suitable fluorinated lipids that may be used in the compositions of thepresent invention also include the fluorinated compounds described inU.S. application Ser. No. 08/887,215 filed Jul. 2, 1997, the disclosureof which is hereby incorporated by reference herein in its entirety,which include the following compounds of formula (IV), formula (V),formula (VI), formula (VII), formula (VIII), formula (IX) and formula(X).

The fluorinated lipid may be a fluorinated fatty acyl derivative, suchas, for example, that of formula (IV):

    CF.sub.3 --(CF.sub.2).sub.n --(CH.sub.2).sub.m --C(═O)--OH(IV)

where n is an integer of from about 7 to about 13, preferably from about9 to about 11; and m is an integer of from 1 to about 4, preferably 1 toabout 2.

The fluorinated lipid be a fluorinated surfactant, such as, for example,a PEG Telomer type compound of formula (V):

    C.sub.x F.sub.2x+1 --(CH.sub.2).sub.z --(OCH.sub.2 CH.sub.2).sub.z --OH(V)

where x is an integer of from about 6 to about 12, preferably from about8 to about 10, more preferably about 9; and z is an integer of fromabout 8 to about 20; preferably from about 8 to about 16; still morepreferably from about 8 to about 12; even more preferably about 8 toabout 10; most preferably about 9.

Further, the fluorinated lipid may be a fluorinated carbohydratederivative, such as, for example, that of formula (VI):

    C.sub.x F.sub.2x+1 --(CH.sub.2).sub.2 --(OCH.sub.2 CH.sub.2).sub.z --O--A(VI)

where x is an integer of from about 6 to about 12; preferably from about8 to about 10; more preferably 9; z is an integer of from about 8 toabout 20; preferably from about 8 to about 16; more preferably fromabout 8 to about 12; still more preferably from about 8 to about 10;most preferably about 9; and A is a monosaccharide or a disaccharide.Suitable monosaccharides and disaccharides include, for example, allose,altrose, glucose, dextrose, mannose, glycerose, gulose, idose,galactose, talose, fructose, psicose, sorbose, rhamnose, tagatose,ribose, arabinose, xylose, lyxose, ribulose, xylulose, erythrose,threose, erythrulose, fucose, sucrose, lactose, maltose, isomaltose,trehalose, cellobiose and the like. Preferably, the monosaccharide ordisaccharide is glucose, dextrose, fructose, mannose, galactose,glucosamine, galactosamine, maltose, sucrose or lactose.

The fluorinated lipid may also be a fluorinated lipophilic derivative,such as, for example, that of formula (VII), which includes thecompounds described in U.S. application Ser. No. 08/465,868, filed Jun.6, 1995, the disclosure of which is hereby incorporated by referenceherein in its entirety: ##STR4## where each of x, y and z isindependently 0 or 1; each X₁ is independently --O--, --S--, --SO--,--SO₂ --, --NR₄ --, --C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)--,--C(═X₂)--NR₄ -- or --NR₄ --C(═X₂)--; X₂ is O or S; Y is a direct bondor --X₃ --M(═O)(OR₅)_(q) --O--, where q is 1 or 2; X₃ is a direct bondor --O--; M is P or S; Z is hydrogen, the residue of a hydrophilicpolymer, a saccharide residue or --N(R₆)_(r), where r is 2 or 3; each R₁is independently an alkyl group of 1 to about 30 carbon atoms or afluorinated alkyl group of 1 to about 30 carbon atoms; R₂ is a directbond or an alkylene linking group of 1 to about 10 carbon atoms; R₃ is adirect bond or an alkylene diradical of 1 to about 10 carbon atoms; eachof R₄ and R₅ is independently hydrogen or an alkyl group of 1 to about 8carbon atoms; and each R₆ is independently hydrogen, an alkyl group of 1to about 8 carbon atoms or a residue of a hydrophilic polymer; providedthat at least one of x, y and z is 1, at least one of R₁ is afluorinated alkyl group of 1 to about 30 carbon atoms; provided thatwhen R₂ is a direct bond, two of x, y and z are each 0.

In formula (VII), each of x, y and z is independently 0 or 1, providedthat at least one of x, y and z is 1. In some embodiments, two of x, yand z are each 0. In other embodiments, one of x, y and z is 0 or 1 andthe other two of x, y and z are each 1, with one of x, y and z being 0and the other two of x, y and z being 1 being more preferred. In otherembodiments, each of x, y and z is 1.

Each X₁ is independently --O--, --S--, --SO--, --SO₂ --, --NR₄ --,--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)--, --C(═X₂)--NR₄ -- or --NR₄--C(═X₂)--. Preferably, each X₁ is independently --O--, --S--,--C(═X₂)--, --C(═X₂)--O--, --O--C(═X₂)--, --C(═X₂)--NR₄ -- or --NR₄--C(═X₂)--. More preferably, each X₁ is independently --C(═X₂)--O-- or--O--C(═X₂)--, most preferably --C(═X₂)--O--.

Each X₂ is O or S, preferably O.

Y is a direct bond or --X₃ --M(═O)(OR₅)_(q) --O--, where q is 1 or 2.Preferably, Y is --X₃ --M(═O)(OR₅)_(q) --O--. M is P or S, preferably P.X₃ is a direct bond or --O--, preferably, a direct bond.

Z is hydrogen atom, the residue of a hydrophilic polymer, a saccharideresidue or --N(R₆)_(r), where r is 2 or 3. In preferred embodiments, Zis --N(R₆)_(r).

Each R₁ is independently an alkyl group of 1 to about 30 carbon atoms ora fluorinated alkyl group of 1 to about 30 carbon atoms, provided thatat least one of R₁ is a fluorinated alkyl group of 1 to about 30 carbonatoms. Thus, when only one of x, y and z is 1, R₁ is necessarily afluorinated alkyl group of 1 to about 30 carbon atoms. In preferredembodiments, where one or none of x, y and z is 0, and preferably whereone of x, y and z is 0 and the other two of x, y and z are each 1, atleast one of R₁ is an alkyl group of 1 to about 30 carbon atoms and atleast one of R₁ is a fluorinated alkyl group of 1 to about 30 carbonatoms. In other embodiments, each R₁ is independently a fluorinatedalkyl group of 1 to about 30 carbon atoms. When a fluorinated alkylgroup of 1 to about 30 carbon atoms, R₁ is preferably a polyfluorinatedalkyl group of 1 to about 30 carbon atoms, with a perfluorinated alkylgroup of 1 to about 30 carbon atoms being more preferred. When afluorinated alkyl group of 1 to about 30 carbon atoms, R₁ is preferablyC_(n) F_(2n+1) --(CH₂)_(m) --, where n is 1 to about 16, preferablyabout 9 to about 14, and m is 0 to about 18, preferably 1 to about 10,more preferably 1 to about 4.

R₂ is a direct bond or an alkylene linking group of 1 to about 10 carbonatoms, provided that when R₂ is a direct bond, two of x, y and z areeach 0. Preferably, R₂ is a direct bond or an alkylene linking group of1 to about 4 carbon atoms. More preferably, R₂ is an alkylene linkinggroup of about 3 carbons. Even more preferably, R₂ is --CH₂ --CH₂ --CH₂--.

R₃ is a direct bond or an alkylene diradical of 1 to about 10 carbons.Preferably, R₃ is a direct bond or an alkylene diradical of 1 to about 4carbon atoms. More preferably, R₃ is an alkylene diradical of about 2carbon atoms. Even more preferably, R₃ is --CH₂ CH₂ --.

Each of R₄ and R₅ is independently a hydrogen atom or an alkyl group of1 to about 8 carbon atoms, preferably of 1 to about 4 carbon atoms. Morepreferably, each of R₄ and R₅ is a hydrogen atom.

R₆ is a hydrogen atom, an alkyl group of 1 to about 8 carbon atoms or aresidue of a hydrophilic polymer. Preferably, R₆ is a hydrogen atom oran alkyl group of 1 to about 4 carbon atoms. More preferably, R₆ is ahydrogen atom or a methyl group, with a methyl group being even morepreferred.

When any symbol appears more than once in a particular formula orsubstituent, such as in formula (VII), its meaning in each instance isindependent of the other, unless otherwise indicated. This independenceof meaning is subject to any of the stated provisos. When each of two ormore adjacent symbols is defined as being "a direct bond" to providemultiple, adjacent direct bonds, the multiple and adjacent direct bondsdevolve into a single direct bond.

Z and R₆ in the definition of Z in formula (VII), can be the residue ofa hydrophilic polymer. Exemplary polymers from which Z and/or R₆ can bederived include polymers in which the repeating units contain one ormore hydroxy groups (polyhydroxy polymers), including, for example,poly(vinyl alcohol); polymers in which the repeating units contain oneor more amino groups (polyamine polymers), including, for example,peptides, polypeptides, proteins and lipoproteins, such as albumin andnatural lipoproteins; polymers in which the repeating units contain oneor more carboxy groups (polycarboxy polymers), including, for example,carboxymethylcellulose, alginic acid and salts thereof, such as sodiumand calcium alginate, glycosaminoglycans and salts thereof, includingsalts of hyaluronic acid, phosphorylated and sulfonated derivatives ofcarbohydrates, genetic material, such as interleukin-2 and interferon,and phosphorothioate oligomers; and polymers in which the repeatingunits contain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates. The molecular weight of thepolymers from which Z and/or R₆ are derived may vary, and is generallyabout 50 to about 5,000,000, with polymers having a molecular weight ofabout 100 to about 50,000 being preferred. More preferred polymers havea molecular weight of about 150 to about 10,000, with molecular weightsof 200 to about 8,000 being even more preferred.

Preferred polymers from which Z and/or R₆ are derived include, forexample, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine),polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymersbeing particularly preferred. Preferred among the PEG polymers are PEGpolymers having a molecular weight of from about 100 to about 10,000.More preferably, the PEG polymers have a molecular weight of from about200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which havemolecular weights of 2,000, 5,000 and 8,000, respectively, being evenmore preferred. Other suitable hydrophilic polymers will be apparent toone skilled in the art in view of the present disclosure. Generally,polymers from which Z and/or R₆ are derived include polymers that can beincorporated in the fluorinated lipids via alkylation or acylationreactions.

As with the various polymers exemplified above, the polymeric residuescan contain functional groups in addition to those typically involved inlinking the polymeric residues to the fluorinated lipids. Suchfunctionalities include, for example, carboxyl, amine, hydroxy and thiolgroups. These functional groups on the polymeric residues can be furtherreacted, if desired, with materials which are generally reactive withsuch functional groups and which can assist in targeting specifictissues in the body including, for example, diseased tissue. Exemplarymaterials which can be reacted with the additional functional groupsinclude, for example, proteins, including antibodies, carbohydrates,peptides, glycopeptides, glycolipids, lectins and nucleosides.

In addition to residues of hydrophilic polymers, Z in formula (VII) canbe a saccharide residue. Exemplary saccharides from which Z can bederived include, for example, monosaccharides or sugar alcohols, such aserythrose, threose, ribose, arabinose, xylose, lyxose, fructose,sorbitol, mannitol and sedoheptulose, with preferred monosaccharidesbeing fructose, mannose, xylose, arabinose, mannitol and sorbitol; anddisaccharides, such as lactose, sucrose, maltose and cellobiose. Othersaccharides include, for example, inositol and ganglioside head groups.Other suitable saccharides, in addition to those exemplified above, willbe readily apparent to one skilled in the art based on the presentdisclosure. Generally, saccharides from which Z is derived includesaccharides that can be incorporated in the fluorinated lipids viaalkylation or acylation reactions.

Preferred fluorinated lipids that are within the scope of formula (VII)are the fluorinated compounds of the formula (VIIa): ##STR5## where n isan integer of from about 7 to about 13, preferably from about 9 to about11; and m is an integer of from about 1 to about 4, preferably 1 toabout 2.

The fluorinated compound may also be a compound of formula (VIII):##STR6## where R₁, R₂, R₃, X₁, Y, Z, x, y and z are as defined informula (VII), including the preferred embodiments thereof, and where eis an integer of from 1 to about 30, preferably about 3 to about 20,more preferably about 4 to about 16, still more preferably about 4 toabout 12, most preferably about 7 to about 9.

In a preferred embodiment, the compound of formula (VIII) may be acompound of the formula (VIIIa): ##STR7## where n and m are as definedabove in formula (VIIa) and where e is as defined above in formula(VIII).

Additionally, the fluorinated compound may be a fluorinated fatty acylderivative, such as, for example, that of formula (IX):

    CF.sub.3 --J--T--C(═O)--OH                             (IX)

Still further, the fluorinated compound may be a fluorinated lipophilicderivative, such as, for example, that of formula (X): ##STR8## In theabove formulas (IX) and (X), J is (--(C═C)_(p1) --(CF₂)_(p2)--(C═C)_(p3) --(CF₂)_(p4) --(C═C)_(p5) --(CF₂)_(p6) --(C═C)_(p7)--(CF₂)_(p8) --(C═C)_(p9) --(CF₂)_(p10) --(C═C)_(p11) --(CF₂)_(p12)--(C═C)_(p13) --), where p1, p2, p3, p4, p5, p6, p7, p8, p9, p10, p11,p12 and p13 are independently an integer of 0, 1 or 2; providedthatthesum of (p1+p2+p3+p4+p5+p6+p7+p8+p9+p10+p11+p12+p13) is an integer offrom about 7 to about 13, and provided that at least one of p2, p4, p6,p8, p10 or p12 is an integer of at least 1; and where T is --(C═C)_(t1)--(CH₂)_(t2) --(C═C)_(t3) --(CH₂)_(t4) --, where t1, t2, t3, and t4 areindependently an integer of 0, 1 or 2; provided that the sum of(t1+t2+t3+t4) is an integer of from 1 to about 4.

Other suitable fluorinated compounds for use as the stabilizingmaterials and/or vesicles of the present invention are described in U.S.Pat. No. 5,562,893, the disclosure of which is hereby incorporatedherein by reference in its entirety. For example, synthetic organicmonomeric repeating units may be used to form polymers suitable asstabilizing materials, including hydroxyacids, lactones, lactides,glycolides, acryl containing compounds, aminotriazol, orthoesters,anyhdrides, ester imides, imides, acetals, urethanes, vinyl alcohols,enolketones, and organosiloxanes.

The method of introducing fluorine into any of these materials is knownin the art. For example, the introduction of perfluoro-t-butyl moietiesis described in U.S. Pat. No. 5,234,680, the disclosure of which ishereby incorporated by reference herein in its entirety. These methodsgenerally involve the reaction of perfluoroalkyl carbanions with hostmolecules, such as (CF₃)₃ C⁻ +R--X→(CF₃)₃ C--R, where R is a hostmolecule and X is a good leaving group, such as bromine, chlorine,iodine or a sulfonato group. After adding a leaving group to theforegoing stabilizing material using methods well known in the art,perfluoro-t-butyl moieties can then be easily introduced to thesederivatized stabilizing materials as described above. Additional methodsare known in the art for the introduction of trifluoromethyl groups intovarious organic compounds. For example, trifluoromethyl groups may beintroduced by nucleophilic perfluoroalkylation usingperfluoroalkyl-trialkylsilanes. Fluorine can be introduced into any ofthe aforementioned stabilizing materials or vesicles either in theirmonomeric or polymeric form. Preferably, fluorine moieties areintroduced into monomers, such as fatty acids, amino acids orpolymerizable synthetic organic compounds, which are then polymerizedfor subsequent use as stabilizing materials and/or vesicles.

The introduction of fluorine into stabilizing materials and/or vesiclesmay also be accomplished by forming vesicles in the presence of aperfluorocarbon gas. For example, when vesicles are formed fromproteins, such as human serum albumin in the presence of aperfluorocarbon gas, such as perfluoropropane, using mechanicalcavitation, fluorine from the gas phase becomes bound to the proteinvesicles during formation. The presence of fluorine in the vesiclesand/or stabilizing materials can be detected by NMR of vesicle debriswhich has been purified from disrupted vesicles. Fluorine can also beintroduced into stabilizing materials and/or vesicles using othermethods, such as sonication, spray-drying or emulsification techniques.

Another way in which fluorine can be introduced into the stabilizingmaterial and/or vesicle is by using a fluorine-containing reactivecompound. The term "reactive compound" refers to compounds which arecapable of interacting with the stabilizing material and/or vesicle insuch a manner that fluorine moieties become covalently attached to thestabilizing material and/or vesicle. When the stabilizing material is aprotein, preferred reactive compounds are either alkyl esters or acylhalides which are capable of reacting with the protein's amino groups toform an amide linkage via an acylation reaction. The reactive compoundcan be introduced at any stage during vesicle formation, but ispreferably added to the gas phase prior to vesicle formation. Forexample, when vesicles are to be made using mechanical or ultrasoundcavitation techniques, the reactive compound can be added to the gasphase by bubbling the gas to be used in the formation of the vesicles(starting gas) through a solution of the reactive compound into the gasphase. The resultant gas mixture, which now contains the starting gasand the reactive compound, is then used to form vesicles. The vesiclesare preferably formed by sonication of human serum albumin in thepresence of a gas mixture, as described in U.S. Pat. No. 4,957,656, thedisclosure of which is hereby incorporated herein by reference in itsentirety.

Suitable fluorine containing alkyl esters and acyl halides for use asstabilizing materials and/or vesicle forming materials in the presentinvention include, for example, diethyl hexafluoroglutarate, diethyltetrafluorosuccinate, methyl heptafluorobutyrate, ethylheptafluorobutyrate, ethyl pentafluoropropionate, methylpentafluoropropionate, ethyl perfluorooctanoate, methylperfluorooctanoate, nonafluoropentanoyl chloride, perfluoropropionylchloride, hexafluoroglutaryl chloride and heptafluorobutyiyl chloride.

Other fluorine containing reactive compound can also be synthesized andused as the stabilizing materials and/or vesicle forming materials inthe present invention, including, for example, aldehydes, isocyanates,isothiocyanates, epoxides, sulfonyl halides, anhydrides, acid halidesand alkyl sulfonates, which contain perfluorocarbon moieties, including--CF₃, --C₂ F₅, --C₃ F₄ and --C(CF₃)₃. These reactive compounds can beused to introduce fluorine moieties into any of the above stabilizingmaterials by choosing a combination which is appropriate to achievecovalent attachment of the fluorine moiety.

Sufficient fluorine should be introduced to decrease the permeability ofthe vesicle to the aqueous environment. This will result in a slowerrate of gas exchange with the aqueous environment which is evidenced byenhanced pressure resistance. Although the specific amount of fluorinenecessary to stabilize the vesicle will depend on the components of thevesicle and the gas contained therein, after introduction of fluorinethe vesicle will preferably contain 0.01 to 20% by weight, and morepreferably about 1.0 to 10% by weight fluorine.

Additionally, oils and fluorinated oils may be used as stabilizingmaterials and/or to stabilize the compositions of the present invention.Suitable oils include, for example, soybean oil, peanut oil, canola oil,olive oil, safflower oil, corn oil, almond oil, cottonseed oil, ethyloleate, isopropyl myristate, isopropyl palnitate, mineral oil, myristylalcohol, octyldodecanol, persic oil, sesame oil, squalene, myristyloleate, cetyl oleate, myristyl palmitate, or any other known ingestibleoil. The oils described herein may be fluorinated, such as triolein witha fluorine (F₂) gas. A "fluorinated oil" refers to an oil in which atleast one hydrogen atom of the oil is replaced with a fluorine atom.Preferably, at least two or more of the hydrogen atoms in the oil arereplaced with fluorine atoms. Other suitable fluorinated oils aredescribed, for example, in U.S. Pat. No. 5,344,930, the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

The stability of vesicles may be attributable, at least in part, to thematerials from which the vesicles are made, and it is often notnecessary to employ additional stabilizing materials, although it isoptional and may be preferred to do so. In addition to the lipidsdiscussed above, the compositions described herein may comprise one ormore other stabilizing materials. Exemplary stabilizing materialsinclude, for example, surfactants, fluorosurfactants and polymers. Thestabilizing materials may be employed to assist in the formation ofvesicles and/or to assure substantial encapsulation of the gases,gaseous precursors and/or bioactive agents. Even for relativelyinsoluble, non-diffusible gases, such as perfluoropropane or sulfurhexafluoride, improved vesicle compositions may be obtained when one ormore stabilizing materials are utilized in the formation of the gasand/or gaseous precursor filled vesicles. These compounds may helpimprove the stability and the integrity of the vesicles with regard totheir size, shape and/or other attributes. Suitable surfactants andfluorosurfactants useful as stabilizing materials for preparing the gasand/or gaseous precursor filled vesicles include the surfactants andfluorosurfactants described in detail herein.

Polymers useful to stabilize the vesicles of the present invention maybe of natural, semi-synthetic (modified natural) or synthetic origin.Suitable natural polymers include naturally occurring polysaccharides,such as, for example, arabinans, fructans, fucans, galactans,galacturonans, glucans, mannans, xylans (such as, for example, inulin),levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins,including amylose, pullulan, glycogen, amylopectin, cellulose, dextran,dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin,agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid,xanthin gum, starch and various other natural homopolyner orheteropolymers, such as those containing one or more of the followingaldoses, ketoses, acids or amines: erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose,gulose, idose, galactose, talose, erythrulose, ribulose, xylulose,psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose,sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaricacid, galacturonic acid, mannuronic acid, glucosamine, galactosamine,and neuraminic acid, and naturally occurring derivatives thereofAccordingly, suitable polymers include, for example, proteins, such asalbumin, polyalginates, and polylactide-coglycolide polymers. Exemplarysemi-synthetic polymers include carboxymethylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose,and methoxycellulose. Exemplary synthetic polymers includepolyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes(such as, for example, polyethylene glycol (including for example, theclass of compounds referred to as Pluronics®, commercially availablefrom BASF, Parsippany, N.J.), polyoxyethylene, and polyethyleneterephthlate), polypropylenes (such as, for example, polypropyleneglycol), polyurethanes (such as, for example, polyvinyl alcohol (.PVA),polyvinyl chloride and polyvinylpyrrolidone), polyamides includingnylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof. Methods for thepreparation of vesicles which employ polymers to stabilize vesiclecompositions will be readily apparent to one skilled in the art, in viewof the present disclosure, when coupled with information known in theart, such as that described and referred to in Unger, U.S. Pat. No.5,205,290, the disclosure of which is hereby incorporated by referenceherein in its entirety.

The compositions of the present invention may be modified and furtherstabilized, for example, by the addition of one or more of a widevariety of (i) viscosity modifiers, including, for example,carbohydrates and their phosphorylated and sulfonal:ed derivatives;polyethers, preferably with molecular weight ranges between 400 and100,000; and di- and trihydroxy alkanes and their polymers, preferablywith molecular weight ranges between 200 and 50,000; (ii) emulsifyingand/or solubilizing agents including, for example, acacia, cholesterol,diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, mono-and di-glycerides, mono-ethanolamine, oleic acid, oleyl alcohol,poloxamer, for example, poloxamer 188, poloxamer 184, and poloxamer 181,Pluronics® (BASF, Parsippany, N.J.), polyoxyethylene 50 stearate,polyoxyl 35 castor oil, polyoxl 10 oleyl ether, polyoxyl 20 cetostearylether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate60, polysorbate 80, propylene glycol diacetate, propylene glycolmonostearate, sodium lauryl sulfate, sodium stearate, sorbitanmonolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitanmonostearate, stearic acid, trolamine, and emulsifying wax; (iii)suspending and/or viscosity-increasing agents, including, for example,acacia, agar, alginic acid, aluminum monostearate, bentonite, magma,carbomer 934P, carboxymethylcellulose, calcium and sodium and sodium 12,carrageenan, cellulose, dextran, gelatin, guar gum, locust bean gum,veegum, hydroxyethyl cellulose, hydroxypropyl methylcellulose,magnesium-aluminum-silicate, Zeolites®, methylcellulose, pectin,polyethylene oxide, povidone, propylene glycol alginate, silicondioxide, sodium alginate, tragacanth, xanthin gum, α-d-gluconolactone,glycerol and mannitol; (iv) synthetic suspending agents, such aspolyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinylalcohol(PVA), polypropylene glycol (PPG), and polysorbate; and (v) tonicityraising agents which stabilize and add tonicity, including, for example,sorbitol, mannitol, trehalose, sucrose, propylene glycol and glycerol.

The compositions of the present invention are desirably formulated in anaqueous environment which can induce the lipids, because of theirhydrophobichydrophilic nature, to form vesicles, which may be the moststable configuration which can be achieved in such an environment.Diluents which may be used to create such an aqueous environmentinclude, for example, water, normal saline, physiological saline,deionized water and water containing one or more dissolved solutes, suchas salts or sugars.

The present stabilizing materials or compositions preferably comprise agas, such as an inert gas. The gas provides the stabilizing materials orcompositions with enhanced reflectivity, particularly in connection withstabilizing materials or compositions in which the gas is entrappedwithin the stabilizing materials or compositions. This may increasetheir effectiveness as drug delivery vehicles or contrast agents.

Preferred gases are inert and biocompatible, and include, for example,air, noble gases, such as helium, rubidium hyperpolarized xenon,hyperpolarized argon, hyperpolarized helium, neon, argon and xenon,carbon dioxide, nitrogen, fluorine, oxygen, sulfur-based gases, such assulfur hexafluoride and sulfur tetrafluoride, fluorinated gases,including, for example, partially fluorinated gases or completelyfluorinated gases, and mixtures thereof. Paramagnetic gases, such as ¹⁷O₂ may also be used in the stabilizing materials and vesicles. It mayalso be desirable to incorporate a precursor to a gaseous substance inthe stabilizing materials or compositions. Such gaseous precursorsinclude materials that are capable of being converted to a gas in vivo,preferably where the gaseous precursor and gas produced arebiocompatible.

Preferably, the gaseous precursor materials comprise compounds that aresensitive to changes in temperature. Exemplary of suitable gaseousprecursors which are sensitive to changes in temperature are theperfluorocarbons and perfluoro ethers. As the artisan will appreciate, aparticular perfluorocarbon or perfluoro ether may exist in the liquidstate when the stabilizing materials are first made, and are thus usedas a gaseous precursor. Alternatively, the perfluorocarbon or perfluoroether may exist in the gaseous state when the stabilizing materials aremade, and are thus used directly as a gas. Whether the perfluorocarbonor perfluoro ether is used as a liquid or a gas generally depends on itsliquid/gas phase transition temperature, or boiling point. For example,a preferred perfluorocarbon, perfluoropentane, has a liquid/gas phasetransition temperature (boiling point) of 29.5° C. This means thatperfluoropentane is generally a liquid at room temperature (about 25°C.), but is converted to a gas within the human body, the normaltemperature of which is about 37° C., which is above the transitiontemperature of perfluoropentane. Thus, under normal circumstances,perfluoropentane is a gaseous precursor. As known to one of ordinaryskill in the art, the effective boiling point of a, substance may berelated to the pressure to which that substance is exposed. Thisrelationship is exemplified by the ideal gas law: PV=nRT, where P ispressure, V is volume, n is moles of substance, R is the gas constant,and T is temperature. The ideal gas law indicates that as pressureincreases, the effective boiling point increases also. Conversely, aspressure decreases, the effective boiling point decreases.

A wide variety of materials can be used as liquids, gases and gaseousprecursors in combination with the stabilizing materials andcompositions of the present invention. For gaseous precursors, it isonly required that the material be capable of undergoing a phasetransition to the gas phase upon passing through the appropriatetemperature. Suitable liquids, gases and/or gaseous precursors for usein the present invention include, for example, hexafluoroacetone,1,3-dichlorotetrafluoroacetone, tetrafluoroallene, boron trifluoride,1,2,3-trichloro-2-fluoro-1,3-butadiene, hexafluoro-1,3-butadiene,1-fluorobutane, perfluorobutane, decafluorobutane, perfluoro-1-butene,perfluoro-2-butene, 2-chloro-1,1,1,4,4,4-hexafluorobutyne,2-chloro-1,1,1,4,4,4-hexafluoro-2-butene, perfluoro-2-butyne,octafluorocyclobutane, perfluorocyclobutene., perfluorocyclobutane,perfluorocyclopentane, octafluorocyclopentene, perfluorocyclopropane,1,1,1-trifluorodiazoethane, hexafluorodimethylamine, perfluoroethane,perfluoropropane, perfluoropentane, hexafluoroethane,hexafluoropropylene, 1,1,2,2,3,3,4,4-octafluorobutane,1,1,1,3,3-pentafluorobutane, octafluoropropane, octafluorocyclopentene,1,1-dichlorofluoroethane, hexafluoro-2-butyne, octafluoro-2-butene,hexafluorobuta-1,3-diene, perfluorodimethylamine,4-methyl-1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane,1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane,1,1,1-trichloro-2,2,2-trifluoroethane,1,1-dichloro-1,2-difluoroethylene,1,1-dichloro-1,2,2,2-tetrafluoroethane,1-chloro-1,1,2,2,2-pentafluoroethane, 1,1-difluoro-2-chloroethane,1,1-dichloro-2-fluoroethane, dichloro-1,1,2,2-tetrafluoroethane,1-chloro-1,1,2,2-tetrafluoroethane, 2-chloro-1,1-difluoroethane,1,1,2-trifluoro-2-chloroethane, 1,2-difluorochloroethane,chloropentafluoroethane, dichlorotrifluoroethane, fluoroethane,nitropentafluoroethane, nitrosopentafluoroethane, perfluoroethylamine,1,2-dichloro-2,2-difluoroethane, 1,1-dichloro-1,2-difluoroethane,1,2-dichloro-1,1,3-trifluoropropane, 1,2-difluoroethane,1,2-difluoroethylene, trifluoromethanesulfonylchloride,trifluoromethanesulfenylchloride, (pentafluorothio)trifluoromethane,trifluoromethanesulfonylfluoride, bromodifluoronitroso-methane,bromofluoromethane, bromochlorodifluoromethane,bromochlorofluoromethane, bromotrifluoromethane, bromotrifluoroethane,chlorodifluoronitromethane, chlorofluoromethane, chlorotrifluoromethane,chlorodifluoromethane, dibromofluoromethane, dibromodifluoromethane,dichlorodifluoromethane, dichlorofluoromethane, 1-bromoperfluorobutane,difluoromethane, difluoroiodomethane, fluoromethane, perfluoromethane,iodotrifluoromethane, iodotrifluoroethylene, nitrotrifluoromethane,nitrosotrifluoromethane, tetrafluoromethane, trichlorofluoromethane,trifluoromethane, perfluoropent-1-ene, 1,1,1,2,2,3-hexafluoropropane,2,2-difluoropropane, heptafluoro-1-nitropropane,heptafluoro-1-nitrosopropane, heptafluoro-2-iodopropane,perfluoropropane, hexafluoropropane,1,1,1,2,3,3-hexafluoro-2,3-dichloropropane,1-bromo-1,1,2,3,3,3-hexafluoropropane, 1-bromoperfluoropropane,2-chloropentafluoro-1,3-butadiene, 3-fluoropropane, 3-fluoropropylene,perfluoropropylene, perfluorotetrahydropyran,perfluoromethyltetrahydrofuran, perfluorobutylmethyl ether,perfluoromethyl-n-butyl ether, perfluoromethylisopropyl ether,perfluoromethyl-t-butyl ether, perfluorobutyl ethyl ether,perfluoromethylpentyl ether, 3,3,3-trifluoropropyne, 3-fluorostyrene,sulfur (di)-decafluoride (S₂ F₁₀), sulfur hexafluoride, seleniumhexafluoride, trifluoroacetonitrile, trifluoromethyl peroxide,trifluoromethyl sulfide, tungsten hexafluoride, 1-bromononafluorobutane,1-chloro-1-fluoro-1-bromomethane, 1-bromo-2,4-difluorobenzene,2-iodo-1,1,1-trifluoroethane, bromine pentafluoride,perfluoro-2-methyl-2-pentene, 1,1,1,3,3-pentafluoropentane,3-fluorobenzaldehyde, 2-fluoro-5-nitrotoluene, 3-fluorostyrene,3,5-difluoroaniline, 2,2,2-trifluoroethylacrylate,3-(trifluoromethoxy)-acetophenone, bis(perfluoroisopropyl) ether,bis(perfluoropropyl) ether, perfluoro isobutyl methyl ether, perfluoron-propyl ethyl ether, perfluoro cyclobutyl methyl ether, perfluorocyclopropyl ethyl ether, perfluoro isopropyl methyl ether, perfluoron-propyl methyl ether, perfluorodiethyl ether, perfluoro cyclopropylmethyl ether, perfluoro methyl ethyl ether, perfluoro dimethyl ether,air, noble gases, such as helium, rubidium hyperpolarized xenon,hyperpolarized argon, hyperpolarized helium, neon, argon, xenon, carbondioxide, nitrogen, isopropyl acetylene, allene, 1,2-butadiere,2,3-butadiene, 1,3-butadiene, 2-methyl-1,3-butadiene, butadiene,2-methylbutane, 1-butene, 2-butene, 2-methyl-1-butene,3-methyl-1-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne,butyl nitrate, 1-butyne, 2-butyne, 3-methyl-1-butyne,2-bromobutyraldehyde, carbonyl sulfide, crotononitrile, cyclobutane,methylcyclobutane, cyclopropane, 3-chlorocyclopentene, dimethylamine,1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane,1,2-dimethylcyclopropane, ethylcyclopropane, methylcyclopropane,diacetylene, 3-ethyl-3-methyl diaziridine, dimethylethylamine,bis(dimethylphosphine)amine, dimethyloxonium chloride,2,3-dimethyl-2-norbomane, 1,3-dioxolane-2-one, 1,1-dichloroethane,1,1-dichloroethylene, chloroethane, 1,1-dichloroethane, methane,chlorodinitromethane, iodomethane, disilanomethane, 2-methylbutane,metbyl ether, methyl isopropyl ether, methyllactate, methylnitrite,methylsulfide, methyl vinyl ether, neon, neopentane, nitrogen, nitrousoxide, 1,2,3-nonadecanetricarboxylic acid 2-hydroxytrimethyl ester,1-nonene-3-yne, 1,4-pentadiene, n-pentane, 4-amino-4-methylpentan-2-one,1-pentene, 2-pentene (cis and trans), 3-bromopent-1-ene,2-chloropropane, tetrachlorophthalic acid, 2,3,6-trimethyl-piperidine,propane, 1-chloropropane, 1-chloropropylene, chloropropylene-(trans),chloropropane-(trans), 2-chloropropylene, 2-aminopropane,1,2-epoxypropane, propene, propyne, 2,4-diaminotoluene, vinyl acetylene,vinyl ether, ethyl vinyl ether, 5-bromovaleryl chloride, 1-bromoethane,6-bromo-1-hexene, 2-bromo-2-nitropropane, 2-bromo-5-nitrothiopbene,2-bromopropene, 3-chloro-5,5-dimethyl-2-cylohexene,2-chloro-2-methylpropane and mixtures thereof. One skilled in the artcould determine whether any compound is a gas, a gaseous precursor or aliquid at any given temperature, in view of the present disclosure.

Preferably, the gas, gaseous precursor and/or liquid compound isperfluoropropane, perfluorobutane, 1-chloro-1-fluoro-1-bromomethane;1,1,1-trichloro-2,2,2-trifluoroethane; 1,2-dichloro-2,2-difluoroethane;1,1-dichloro-1,2-difluoroethane; 1,2-dichloro-1,1,3-trifluoropropane;1,1,2,2,3,3,4,4-octafluorobutane; 1,1,1,3,3-pentafluorobutane;1-bromoperfluorobutane; perfluorocyclohexane;1-bromo-2,4-difluorobenzene; 2-iodo-1,1,1-trifluoroethane;5-bromovaleryl chloride; 1,3-dichlorotetrafluoroacetone; brominepentafluoride; 1-bromo-1,1,2,3,3,3 -hexafluoropropane;2-chloro-1,1,1,4,4,4-hexafluoro-2-butene;2-chloropentafluoro-1,3-butadiene; iodotrifluoroethylene;1,1,2-trifluoro-2-chloroethane; 1,2-difluorochloroethane;1,1-difluoro-2-chloroethane; 1,1-dichloroflouroethane;heptafluoro-2-iodopropane; 3-fluorobenzaldehyde;2-fluoro-5-nitrotoluene; 3-fluorostyrene; perfluoro-2-methyl-2-pentene;3,5-difluoroaniline; 2,2,2-trifluoroethylacrylate;3-(trifluoromethoxy)-acetophenone; 1,1,2,2,3,3,4,4-octafluorobutane;1,1,1,3,3-pentafluorobutane; perfluorocyclohexane;perfluoromethyl-n-butyl either; perfluoromethylisopropyl ether;perfluoromethyl-t-butyl ether; 1-fluorobutane; 1-bromoethane;6-bromo-1-hexene; 2-bromo-2-nitropropane; 2-bromo-5-nitrothiophene;2-bromopropene; 3-chloro-5,5-dimethyl-2-cyclohexene and2-chloro-2-methyl-propane. Under conditions of shaking or emulsificationfollowed optionally by lyophilization these compounds will partitioninto the internal space of the compositions and becomed entrappedagainst rapid diffusion.

Preferred gases and gaseous precursors are compounds which are sparinglysoluble in water but which may, in some cases, be liposoluble, such aslow molecular weight alkanes and their fluorinated analogs. In preferredembodiments, the gas comprises a fluorinated gas, which includes gasescontaining one or more fluorine atoms. Preferred are gases which containmore than one fluorine atom, with perfluorocarbons (fully fluorinatedfluorocarbons) being more preferred. Preferred gases and gaseousprecursors include, for example, fluorinated carbons, perfluorocarbons,sulfur hexafluoride, perfllioro ethers and combinations thereof.

Preferred perfluorocarbons generally have from 1 to about 4 carbon atomsand from about 4 to about 10 fluorine atoms, most preferablyperfluorobutane (C₄ F₁₀). Preferred gaseous precursors generally havefrom about 4 to about 8 carbon atoms, more preferably about 5 or about 6carbon atoms, and from about 12 to about 15 fluorine atoms. Theperfluorocarbon gas may be saturated, unsaturated or cyclic, including,for example, perfluoromethane, perfluoroethane, perfluoropropane,perfluorocyclopropane, perfluorobutane, perfluorocyclobutane,perfluoropentane, perfluorocylcopentane, and mixtures thereof. Morepreferably, the perfluorocarbon gas is perfluoropentane,perfluoropropane or perfluorobutane, with perfluoropropane beingparticularly preferred.

Preferred ethers for use in the present invention include partially orfully fluorinated ethers, preferably having a boiling point of fromabout 36° C. to about 60° C. Fluorinated ethers are ethers in which oneor more hydrogen atoms is replaced by a fluorine atom. Fluorinatedethers have the general formula CX₃ (CX₂)_(n) --O--(CX₂)_(n) CX₃,wherein X is a hydrogen atom, a fluorine atom or another halogen atomprovided that at least one of X is a fluorine atom. Generally,fluorinated ethers containing about 4 to about 6 carbon atoms will havea boiling point within the preferred range for the invention, althoughsmaller or larger chain fluorinated ethers may also be employed inappropriate circumstances. Preferred fluorinated ethers for use in thepresent invention include, for example, perfluorotetrahydropyran,perfluoromethyltetrahydrofuran, perfluorobutylmethyl ether (e.g.,perfluoro t-butylmethyl ether, perfluoro isobutyl methyl ether,perfluoro n-butyl methyl ether), perfluoropropylethyl ether (e.g.,perfluoro isopropyl ethyl ether, perfluoro n-propyl ethyl ether),perfluorocyclobutylmethyl ether, perfluorocyclopropyl ethyl ether,perfluoropropylmethyl ether (e.g., perfluoro isopropyl methyl ether,perfluoro n-propyl methyl ether), perfluorodiethyl ether,perfluorocyclopropylmethyl ether, perfluoromethylethyl ether andperfluorodimethyl ether.

Other preferred fluoroether compounds contain between 4 and 6 carbonatoms, and optionally contain one halide ion, preferably Br¹⁻. Forexample, compounds having the structure C_(n) F_(y) H_(x) OBr, where nis an integer of from 1 to about 6, y is an integer of from 0 to about13, and x is an integer of from 0 to about 13, are useful as gaseousprecursors. Examples of useful gaseous precursors having this formulainclude perfluoropropyloxylbromide and 2-bromooxyperfluoropropane.

Another preferable gas is sulfur hexafluoride. Yet another preferablegas is heptafluoropropane, including 1,1,1,2,3,3,3-heptafluoropropaneand its isomer, 1,1,2,2,3,3,3-heptafluoropropane. Other compounds thatmay be used as gaseous precursors in the present invention includecompounds comprising a sulfur atom, including compounds of the formulaCF₃ --(CF₂)_(n) --SF₅ or SF₅ --(CF₂)_(n) --SF₅, where n is an integer offrom 1 to about 10. Mixtures of different types of gases, such asmixtures of a perfluorocarbon or a perfluoro ether and another type ofgas, such as, for example, air or nitrogen, can also be used in thecompositions of the present invention. Other gases, including the gasesexemplified above, would be apparent to one skilled in the art in viewof the present disclosure.

Other gaseous precursors which are suitable for use in stabilizingmaterials and compositions described herein are agents which aresensitive to pH. These agents include materials that are capable ofevolving gas, for example, upon being exposed to a pH that is neutral oracidic. Examples of such pH sensitive agents include salts of an acidwhich is selected from the group consisting of inorganic acids, organicacids and mixtures thereof Carbonic acid (H₂ CO₃) is an example of asuitable inorganic acid, and aminomalonic acid is an example of asuitable organic acid. Other acids, including inorganic and organicacids, would be apparent to one skilled in the art in view of thepresent disclosure.

Gaseous precursors derived from salts are preferably selected from thegroup consisting of alkali metal salts, ammonium salts and mixturesthereof More preferably, the salt is selected from the group consistingof carbonate, bicarbonate, sesquecarbonate, aminomalonate and mixturesthereof Suitable gaseous precursor materials which are derived fromsalts include, for example, lithium carbonate, sodium carbonate,potassium carbonate, lithium bicarbonate, sodium bicarbonate, potassiumbicarbonate, magnesium carbonate, calcium carbonate, magnesiumbicarbonate, ammonium carbonate, ammonium bicarbonate, ammoniumsesquecarbonate, sodium sesquecarbonate, sodium aminomalonate andammonium aminomalonate. Aminomalonate is well known in the art, and itspreparation is described, for example, in Thanassi, Biochemistry,9(3):525-532 (1970); Fitzpatrick et al., Inorganic Chemistry,13(3):568-574 (1974); and Stelmashok et al., Koordinatsionnaya Khimiya,3(4):524-527 (1977). The disclosures of each of these publications arehereby incorporated herein by reference in their entirety.

The gaseous precursor materials may be also photoactivated materials,such as a diazonium ion and aminomalonate. As discussed more fullyhereinafter, certain stabilizing materials and/or vesicles, particularlyvesicles, may be formulated so that gas is formed at the target tissueor by the action of sound on the stabilizing materials. Examples ofgaseous precursors are described, for example, in U.S. Pat. Nos.5,088,499 and 5,149,319, the disclosures of each of which are herebyincorporated herein by reference in their entirety. Other gaseousprecursors, in addition to those exemplified above, will be apparent toone skilled in the art in view of the present disclosure.

The gases and/or gaseous precursors are preferably incorporated in thestabilizing materials and/or compositions irrespective of the physicalnature of the composition. Thus, the gases and/or gaseous precursors maybe incorporated, for example, in stabilizing materials in which thestabilizing materials are aggregated randomly, such as emulsions,dispersions or suspensions, as well as in vesicles, including vesiclessuch as cochleates, micelles and liposomes. Incorporation of the gasesand/or gaseous precursors in the stabilizing materials and/orcompositions may be achieved by using any of a number of methods. Forexample, gas filled compositions can be produced by shaking or otherwiseagitating an aqueous mixture which comprises a gas and/or gaseousprecursor and one or more lipids. This promotes the formation ofstabilized compositions within which the gas and/or gaseous precursor isencapsulated.

In addition, a gas may be bubbled directly into an aqueous mixture ofstabilizing materials and/or vesicle-forming compounds. Alternatively, agas instillation method can be used as disclosed, for example, in U.S.Pat. Nos. 5,352,435 and 5,228,446, the disclosures of each of which arehereby incorporated herein by reference in their entirety. Suitablemethods for incorporating the gas and/or gaseous precursor in cationiclipid compositions are disclosed also in U.S. Pat. No. 4,865,836, thedisclosure of which is hereby incorporated herein by reference in itsentirety. Other methods would be apparent to one skilled in the artbased on the present disclosure. Preferably, the gas may be instilled inthe stabilizing materials and/or other compositions after or during theaddition of the stabilizing material and/or during formation ofvesicles.

It is preferred that the stabilizing materials, and especially thevesicles, be formulated from lipids and optional stabilizing compoundsto promote the formation of stable vesicles, as discussed above.Additionally, it is preferred that the stabilizing materials and/orvesicles comprise a highly stable gas as well. The phrase "highly stablegas" refers to a gas which has limited solubility and diffusability inaqueous media. Exemplary highly stable gases include perfluorocarbonssince they are generally less diffusible and relatively insoluble inaqueous media. Accordingly, their use may promote the formation ofhighly stable vesicles.

Compositions employed herein may also include, with respect to theirpreparation, formation and use, gaseous precursors that can be activatedto change from a liquid or solid into a gas by temperature, pH, light,and energy (such as ultrasound). The gaseous precursors may be made intogas by storing the precursors at reduced pressure. For example, a vialstored under reduced pressure may create a headspace of perfluoropentaneor perfluorohexane gas, useful for creating a preformed gas prior toinjection. Preferably, the gaseous precursors may be activated bytemperature. Set forth below is a table listing a series of gaseousprecursors which undergo phase transitions from liquid to gaseous statesat relatively close to normal body temperature (37° C.) or below, andthe size of the emulsified droplets that would be required to form avesicle of a maximum size of 10 μm.

                  TABLE 1                                                         ______________________________________                                        Physical Characteristics of Gaseous Precursors and                            Diameter of Emulsified Droplet to Form a 10 μm Vesicle                                         Boiling       Diameter (μm) of                                    Molecular                                                                              Point         emulsified droplet to                       Compound   Weight   (° C.)                                                                         Density                                                                             make 10 μm vesicle                       ______________________________________                                        perfluoropentane                                                                         288.04   28.5    1.7326                                                                              2.9                                         1-fluorobutane                                                                           76.11    32.5    0.67789                                                                             1.2                                         2-methyl butane                                                                          72.15    27.8    0.6201                                                                              2.6                                         2-methyl 1-butene                                                                        70.13    31.2    0.6504                                                                              2.5                                         2-methyl-2-butene                                                                        70.13    38.6    0.6623                                                                              2.5                                         1-butene-3-yne-2-                                                                        66.10    34.0    0.6801                                                                              2.4                                         methyl                                                                        3-methyl-1-butyne                                                                        68.12    29.5    0.6660                                                                              2.5                                         octafluorocyclo-                                                                         200.04   -5.8    1.48  2.8                                         butane                                                                        decafluorobutane                                                                         238.04   -2      1.517 3.0                                         hexafluoroethane                                                                         138.01   -78.1   1.607 2.7                                         ______________________________________                                         Source: Chemical Rubber Company Handbook of Chemistry and Physics, Robert     C. Weast and David R. Lide, eds., CRC Press Inc. Boca Raton Florida           (1989-1990)                                                              

As noted above, it is preferred to optimize the utility of thestabilizing materials and/or vesicles, especially vesicles formulatedfrom lipids, by using gases of limited solubility. The phrase "limitedsolubility" refers to the ability of the gas to diffuse out of thevesicles by virtue of its solubility in the surrounding aqueous medium.A greater solubility in the aqueous medium imposes a gradient with thegas in the vesicle such that the gas may have a tendency to diffuse outof the vesicle. A lesser solubility in the aqueous milieu, may, on theother hand, decrease or eliminate the gradient between the vesicle andthe interface such that diffusion of the gas out of the vesicle may beimpeded. Preferably, the gas entrapped in the vesicle has a solubilityless than that of oxygen, that is, about 1 part gas in about 32 partswater. See Matheson Gas Data Book, 1966, Matheson Company Inc. Morepreferably, the gas entrapped in the vesicle possesses a solubility inwater less than that of air; and even more preferably, the gas entrappedin the vesicle possesses a solubility in water less than that ofnitrogen.

In the present invention, the lipids are typically prepared in a mixtureand the counter ion is added to the mixture at any stage in thepreparation of the lipid composition. For example, the counter ion maybe added during one or another agitating procedures including, forexample, shaking, microemulsification and/or sonication. Preferably, thecounter ion is added along with the lipids at the initial stage of thepreparation of the composition. Accordingly, in any of the methodsdescribed throughout the present disclosure, a counter ion may be usedin the preparation of the stabilizing materials, compositions, vesicles,liposomes, gas filled vesicles, gaseous precusor filled vesicles, andthe like. Preferably, in the methods described throughout the presentdisclosure, the counter ion is added along with the lipids at theinitial stage of the preparation of the compositions.

One or more bioactive agents may be incorporated into the lipidcompositions described herein. The bioactive agent may be added to themixture of lipids at the initial stage of preparation of the lipidcomposition, prior to the addition of the counter ion or after thecompositions are formed. For example, in the delivery ofdeoxyribonucleic acid (DNA), preformed compositions, composed ofphosphatidic acid, a lipid covalently bonded to a polymer, and calcium,bind DNA to the compositions. Also the DNA can be incorporated into thecompositions by adding the DNA to the lipids at the same time as thecounter ion is added to the lipid mixture. Relatively small amounts ofenergy are necessary to produce the particles of very small size underthe appropriate conditions (e.g. appropriate concentrations of lipidmaterials).

A wide variety of bioactive agents may be entrapped in the compositionsof the present invention. Suitable bioactive agents include, forexample, contrast agents, genetic materials, chemotherapeutics, peptidesand nucleic acids. The compositions of the present invention may also beused for stabilizing gas bodies for use in ultrasound and drug delivery.Preferably, the bioactive agent is genetic material, which includes, forexample, nucleic acids, RNA and DNA, of either natural or syntheticorigin, including recombinant RNA and DNA and antisense RNA and DNA,hammerhead RNA, ribozymes, hammerhead ribozymes, antigene nucleic acids,both single and double stranded RNA and DNA and analogs thereof,ribooligonucleotides, deoxyribooligonucleotides, antisenseribooligonucleotides, and antisense deoxyribooligonucleotides.

Other bioactive agents that may be used in the compositions of thepresent invention include, for example, LHRH analogs, 5-lipooxygenaseinhibitors, immunosuppressants or bronchodilators; especially preferredmaterials include leuprolide acetate. The LHRHAc-D-2-Nal-D-4-Cl-Phe-D-3-Pal-Ser-N-MeTyr-D-Lys(Nic)-Leu-Lys(N-Isp)-Pro-D-Ala-NH₂(hereinafter "D-2-Nal"), the 5-lipoxygenase inhibitorN-[3-[5-(4-fluorophenylmethyl)-2-thienyl]-1methyl-2-propynyl]-N-hydroxyurea,the immunosuppressant cyclosporin A, and the adrenergic bronchodilatorsisoproterenol arid albuterol. (As used herein, the terms "5-lipoxygenaseinhibitor" or "5-LO inhibitor" refer to any physiologically activecompound capable of affecting leukotriene biosynthesis.)

The compositions of the present invention are also suitable for theadministration of a wide variety of peptide and non-peptide bioactiveagents. Some examples of peptides which may be incorporated into thecompositions are interferons and other macrophage activation factors,such as lymphokines, muramyl dipeptide (MDP), γ-interferon, α-interferonand β-interferon, and related antiviral and tumoricidal agents; opioidpeptides and neuropeptides, such as enkaphalins, endorphins anddynorphins, and related analgesics; renin inhibitors includingnew-generation anti-hypertensive agents; cholecystokinins (CCK analogs)such as CCK, ceruletide and eledoisin, and relatedcardiovascular-targeting agents and CNS-targeting agents; leukotrienesand prostaglandins, such as oxytocin, and related anti-inflammatory,oxytocid and abortifacient compounds; erythropoietin and analogsthereof, as well as related haematinics; LHRH analogs, such asleuprolide, buserelin and nafarelin, and related down-regulators ofpituitary receptors; parathyroid hormone and other growth hormoneanalogs; enzymes, such as Dnase, catalase and alpha-1 antitrypsin;immunosuppressants such as cyclosporin; GM-CSF and otherimmunomodulators; and insulin.

Non-peptides which may be used in the compositions and methods of thepresent invention include, for example, beta-agonists, such asisoproterenol, albuterol, isoetherine and metoproteronol, and relatedanti-asthmatics; steroids, such as flunisolide, and similaranti-asthmatics; cholinergic agents, such as cromolyn, and relatedanti-asthmatics; and 5-lipoxygenase inhibitors, such as zileuton and thehydroxyurea compound described above, and related leukotrieneinhibitors.

Bioactive agents that act as antineoplastics and antibiotics may also bedelivered using the compositions of the present invention. Among theseare included, for example, antibiotics such as p-aminosalicylic acid,isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochlorideethionamide, pyrazinamide, rifampin and streptomycin sulfate, dapsone,chloramphenicol, neomycin, ceflacor, cefadroxil, cephalexin, cephadrineerythromycin, clindamycin, lincomycin, amoxicillin, ampicillin,bacampicillin, carbenicillin, dicloxicillin, cyclacillin, picloxicillin,hetacillin, methicillin, nafcillin, oxacillin, penicillin (G and V),ticarcillin rifampin, tetracycline and amphotericin B; and antitumordrugs such as methotrexate, fluorourcil, adriamycin, mitomycin,ansamitomycin, bleomycin, cystiene arabinoside, arabinosyl adenine,mercaptopolylysine, vincristine, busulfan, chlorambucil, azidothymidine,melphalan (e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine,mitotane, procarbazine hydrochloride dactinomycin (actinomycin D),daunorubicin hydrochloride, doxorubicin hydrochloride, taxol, plicamycin(mithramycin), aminoglutethimide, estramustine phosphate sodium,flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate,testolactone, trilostane, amsacrine (m-AMSA), asparaginase, etoposide(VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, hydroxyurea,procarbazine, and dacarbazine; mitotic inhibitors such as the vincaalkaloids, and steroids such as dexamethasone.

Charged bioactive agents, such as DNA, can be readily incorporated intothe compositions through noncovalent interactions, such as ionic orelectrostatic interactions, between the charged bioactive agent, thecounter ion and the charged lipid. Hydrophobic bioactive agents, such assterols, can be added to the lipid mixture and then when the counter ionis added this can be employed to incorporate the lipophilic bioactiveagent into the core of the noncovalently crosslinked compositions.Bioactive agents, such as peptides, can also be incorporated when theyare hydrophobic, neutral or charged. In many cases by using theappropriate lipids, an interaction can be formed between the solublelipid and peptide or other bioactive agent. For lipophilic drugs, theinteraction may be hydrophobic or van der Waals forces. For chargeddrugs and lipid head groups, the interaction may be electrostaticinteractions. When the counter ion is added, the bioactive agent is thengenerally entrapped within the lipid composition. Additionally, thecounter ions themselves may be employed as the therapeutic agents, suchas, for example, Ca⁺² for the treatment of calcium deficiency.

The compositions of the present invention may also comprise a targetingmoiety, such as a targeting ligand. Targeting ligands are preferablyassociated with the compositions covalently or non-covalently. Thetargeting ligand may be bound, for example, via a covalent ornon-covalent bond, to at least one of the lipids in the composition.Preferably, the targeting ligand is bound to the compositionscovalently. In the case of lipid compositions which comprisecholesterol, the targeting ligand is preferably bound to the cholesterolsubstantially only non-covalently, and/or the targeting ligand is boundcovalently to a component of the composition, for example, anotherlipid, such as a phospholipid, other than the cholesterol.

The targeting ligands which are incorporated in the compositions of thepresent invention are preferably substances which are capable oftargeting receptors and/or tissues in vivo and/or in vitro. With respectto the targeting of tissue, the targeting ligands are desirably capableof targeting heart tissue and membranous tissues, including endothelialand epithelial cells. In the case of receptors, the targeting ligandsare desirably capable of targeting GPIIbIIIa receptors or lymphocytereceptors, such as T-cells, B-cells or interleukin-2 receptors.Preferred targeting ligands for use in targeting tissues and/orreceptors, including the tissues and receptors exemplified above, areselected from the group consisting of proteins, including antibodies,antibody fragments, hormones, hormone analogues, glycoproteins andlectins, peptides, polypeptides, amino acids, sugars, such assaccharides, including monosaccharides and polysaccharides, andcarbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors,and genetic material, including nucleosides, nucleotides, nucleotideacid constructs and polynucleotides, with peptides being particularlypreferred.

An example of a protein which may be preferred for use as a targetingligand is Protein A, which is protein that is produced by most strainsof Staphylococcus aureus. Protein A is commercially available, forexample, from Sigma Chemical Co. (St. Louis, Mo.). Protein A may then beused for binding a variety of IgG antibodies. Generally speaking,peptides which are particularly useful as targeting ligands includenatural, modified natural, or synthetic peptides that incorporateadditional modes of resistance to degradation by vascularly circulatingesterases, amidases, or peptidases. A useful method of stabilization ofpeptide moieties incorporates the use of cyclization techniques. As anexample, the end-to-end cyclization whereby the carboxy terminus iscovalently linked to the amine terminus via an amide bond may be usefulto inhibit peptide degradation and increase circulating half-life.Additionally, a side chain-to-side chain cyclization or end-to-sidechain cyclization is also useful in inducing stability. In addition, thesubstitution of an L-amino acid for a D-amino acid in a strategic regionof the peptide may offer resistance to biological degradation.

Preferred targeting ligands in the present invention include celladhesion molecules (CAM), among which are, for example, cytokines,integrins, cadherins, immunoglobulins and selecting, all of which arediscussed in detail below.

In connection with the targeting of endothelial cells, suitabletargeting ligands include, for example, one or more of the following:growth factors, including, for example, basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), transforming growthfactor-alpha (TGF-α), transforming growth factor-beta (TGF-β),platelet-derived endothelial cell growth factor (PD-ECGF) vascularendothelial growth factor (VEGF) and human growth factor (HGF);angiogenin; tumor necrosis factors, including tumor necrosisfactor-alpha (TNF-α) and tumor necrosis factor-beta (TNF-β), andreceptor antibodies and fragments thereof to tumor necrosis factor (TNF)receptor 1 or 2 family, including, for example, TNF-R1, TNF-R2, FAS,TNFR-RP, NGF-R, CD30, CD40, CD27, OX40 and 4-1BB; copper-containingpolyribo-nucleotide angiotropin with a molecular weight of about 4,500,as well as low molecular weight non-peptide angiogenic factors, such as1-butyryl glycerol; the prostaglandins, including, for example,prostaglandin E₁ (PGE₁) and prostaglandin E₂ (PGE₂); nicotinamide;adenosine; dipyridamole; dobutamine; hyaluronic acid degradationproducts, such as, for example, degradation products resulting fromhydrolysis of β linkages, including hyalobiuronic acid; angiogenesisinhibitors, including, for example, collagenase inhibitors; minocycline;medroxy-progesterone; chitin chemically modified with 6-O-sulfate and6-O-carboxy-methyl groups; angiostatic steroids, such astetrahydrocortisol; and heparin, including fragments of heparin, suchas, for example, fragments having a molecular weight of about 6,000,admixed with steroids, such as, for example, cortisone orhydrocortisone; angiogenesis inhibitors, including angioinhibin(AGM-1470--an angiostatic antibiotic); platelet factor 4; protamine;sulfated polysaccharide peptidoglycan complexes derived from thebacterial wall of an Arthobacter species; fungal-derived angiogenesisinhibitors, such as fumagillin derived from Aspergillus fumigatus;D-penicillamine; gold thiomalate; thrombospondin; vitamin D₃ analogues,including, for example, 1-α, 25-dihydroxyvitamin D₃ and a syntheticanalogue 22-oxa-1-α, 25-dihydroxy-vitamin D₃ ; interferons, including,for example, α-interferon, β-interferon and γ-interferon; cytokines andcytokine fragments, such as the interleukins, including, for example,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3),interleuikin-4 (IL-4), interleukin-5 (IL-5), interluekin-6 (IL-6),interleukin-7 (IL-7) and interleukin-8 (IL-8); erythropoietin; a 20-merpeptide or smaller for binding to receptor or antagonists to nativecytokines; granulocyte macrophage colony stimulating factor (GMCSF);LTB₄ leukocyte receptor antagonists; heparin, including low molecularweight fragments of heparin or analogues of heparin; simple sulfatedpolysaccharides, such as cyclodextrins, including α-cyclodextrin,β-cyclodextrin, and γ-cyclodextrin; tetradecasulfate; transferrin;ferritin; platelet factor 4; protamine; Gly-His-Lys complexed to copper;ceruloplasmin; (12R)-hydroxyeico-satrienoic acid; okadaic acid; lectins;antibodies; CD11a/CD18; and Very Late Activation Integrin-4 (VLA-4).

In another embodiment, small peptides which bind the interluekin-1(IL-1) receptor may be used. For example, peptides generated by phagedisplay core sequences of QPY have been shown to be essential forpeptide binding, including, for example, AF12198, a 15-mer with a coresequence of WYQJY, where J is azetidine; and IL-1 antagonists with K_(d)10⁻¹⁰ to 10⁻¹² M, such as AcPhe-Glu,Trp-Pro-Gly-Trp-Tyr-Gln-Aze-Tyr-Ala-Leu-Pro-Leu-CONH₂ orAc-Phe-Glu-Trp-Pro-Gly-Trp-Tyr-Gln-Aze-Tyr-Ala-Leu-Pro-Leu-.

Endothelial-leukocyte adhesion molecules (ELAM's) are antigens which areexpressed by endothelial cells under conditions of stress which thenfacilitate the migration of the leukocytes across the endothelium liningthe vasculature into the surrounding tissues. These sameendothelial-leukocyte adhesion molecules may also be advantageouslyexploited as receptors for targeting of vesicles. These endothelial celladhesion molecules belong to a family known as selectins in which theknown members, such as GMP-140, all participate in endothelial-leukocyteadhesion and include ELAM-1, LAM-1 and the granule membrane protein 140(GMP-140) also known as platelet activation-dependent granule-externalmembrane protein (PADGEM), VCAM-1/INCAM-110 (Vascular AdhesionMolecule/Inducible Adhesion Molecule) and ICAM-1 (Intercellular AdhesionMolecule).

The cadherin family of cell adhesion molecules may also be used astargeting iligands, including for example, the E-, N-, and P-cadherins,cadherin-4, cadherin-5, cadherin-6, cadherin-7, cadherin-8, cadherin-9,cadherin-10, and cadherin-11; and most preferably cadherin C-5. Further,antibodies directed to cadherins, such as, for example, the monoclonalantibody Ec6C10, may be used to recognize cadherins expressed locally byspecific endothelial cells.

A wide variety of different targeting ligands can be selected to bind tothe cytoplasmic domains of the ELAM molecules. Targeting ligands in thisregard may include lectins, a wide variety of carbohydrate or sugarmoieties, antibodies, antibody fragments, Fab fragments, such as, forexample, Fab'2, and synthetic peptides, including, for example,Arginine-Glycine-Aspartic Acid (R-G-D) which may be targeted to woundhealing. While many of these materials may be derived from naturalsources, some may be synthesized by molecular biological recombinanttechniques and others may be synthetic in origin. Peptides may beprepared by a variety of techniques known in the art. Targeting ligandsderived or modified from human leukocyte origin, such as CD11a/CD18, andleukocyte cell surface glycoprotein (LFA-1), may also be used as theseare known to bind to the endothelial cell receptor ICAM-1. The cytokineinducible member of the immunoglobulin superfamily, VCAM-1, which ismononuclear leukocyte-selective, may also be used as a targeting ligand.VLA-4, derived from human monocytes, may be used to target VCAM-1.Antibodies and other targeting ligands may be employed to targetendoglin, which is an endothelial cell proliferation marker. Endoglin isupregulated on endothelial cells in miscellaneous solid tumors. Atargeting ligand which may be used to target endoglin is the antibodyTEC-11. Thorpe et al, Breast Cancer Research and Treatment, 36:237-51(1995).

Endothelial cell activation in the setting of atherosclerosis is used inthis invention to target the compositions to regions of arteriosclerosisincluding, for example, atherosclerotic plaque. One such target that canbe used is the inducible mononuclear leukocyte endothelial adhesionmolecule recognized by Rb1/9 as an ATHERO-ELAM. The monoclonalantibodies, H4/18 and H18/7, may be used to target endothelial cellsurface antigens which are induced by cytokine mediators. As a preferredembodiment, gas filled compositions are targeted to atheroscleroticplaque to non-invasively detect diseased blood vessels before severedamage has occurred, for example, prior to stroke or myocardialinfarction, so that appropriate medical or surgical intervention may beimplemented. ATHERO-ELAM is a preferred target and ligands, such asantibodies, peptides, or lectins or combinations thereof may be used totarget this cell surface epitope expressed on endothelial cells in thecontext of atherosclerosis. Alternatively, lipoproteins or lipoproteinfragments derived from low or high density lipoprotein proteins may beused as targeting ligands. Additionally, cholesterol may be used totarget the endothelial cells and localize the lipids, vesicles, and thelike, to regions of atherosclerotic plaque. In embodiments which involvethe use of cholesterol as a targeting ligand, the cholesterol ispreferably unmodified (non-derivatized) with other chemical groups,moieties and ligands.

A targeting ligand directed toward thrombotic material in plaque may beused to differentiate between active and inactive regions ofatherosclerotic plaque. Active plaques in the process of generatingthrombi are more dangerous since they may ultimately occlude a vessel orresult in emboli. In this regard, in addition to low molecular weightheparin fragments, other targeting ligands, such as, for example,anti-fibrin antibody, tissue plasminogen activator (t-PA), anti-thrombinantibody and fibrin antibodies directed to platelet activation factions,may be used to target active plaque with evolving clots. Most preferredtargeting ligands are those which will target a plasma membraneassociated GPIIbIIIa in activated platelets in addition to targetingP-selectin, and an antibody or associated antibody fragment directed toGPIIbIIIa. The present invention is also useful for detecting regions ofacute myocardial infarction. By attaching anti-myosin (particularlycardiomyosin) antibody or anti-actin antibodies to the lipids, infarctedmyocardium may be detected by the methods of the present invention. Fortargeting to granulation tissue (healing wounds), many of the abovetargeting ligands may be useful, The wound healing tripeptide,arginine-glycine-aspartic acid (RGD), may also be used is a targetingligand in this regard.

As with the endothelial cells discussed above, a wide variety ofpeptides, proteins and antibodies may be employed as targeting ligandsfor targeting epithelial cells. Preferably, a peptide, includingsynthetic, semi-synthetic or naturally-occurring peptides, with highaffinity to the epithelial cell target receptor may be selected, withsynthetic peptides being more preferred. In connection with thesepreferred embodiments, peptides having from about 5 to about 15 aminoacid residues are preferred. Antibodies may be used as whole antibody orantibody fragments, for example, Fab or Fab'2, either of natural orrecombinant origin. The antibodies of natural origin may be of animal orhuman origin, or may be chimeric (mouse/human). Human recombinant orchimeric antibodies are preferred and fragments are preferred to wholeantibody.

Examples of monoclonal antibodies which may be employed as targetingligands in the present compositions include CALAM 27, which is formed byimmunizing BALEI/c mice with whole human squamous cell carcinoma of thetongue and forming hybridomtas by crossing extracted spleen cells withthose of an NS1 syngeneic myeloma cell line. Gioanni et al, CancerResearch, 47: 4417-4424 (1987). CALAM 27 is directed to surface epitopesof both normal and malignant epithelial cells. Normal lymph nodesgenerally do not contain cells expressing these epitopes. See CancerResearch, 47:4417-4424 (1987). Accordingly, compositions comprising thisantibody can be used to target metastases in the lymph nodes. Themonoclonal antibody 3C2 may be employed as a targeting ligarld fortargeting malignant epithelial cells of serious ovarian carcinoma andendometrioid carcinoma. Another exemplary targeting ligand is Mab 4C7(see Cancer Research, 45:2358-2362 (1985)), which may be used to targetmucinous carcinoma, endometriod carcinoma and mesonephroid carcinoma.For targeting squamous cell carcinoma in head and neck cancer, Mab E48(Biological Abstract, Vol. 099 Issue. 066 Ref 082748) may be used as atargeting ligand. For targeting malignant melanoma, the monoclonalantibody 225.28s (Pathol. Biol., 38 (8):866-869 (1990)) may be employed.The monoclonal antibody mAb2E₁, which is targeted to EPR-1 (effectorcell protease 1), may also be used.

Targeting ligands may be selected for targeting antigens, includingantigens associated with breast cancer, such as epidermal growth factorreceptor (EGFR), fibroblast growth factor receptor, erbB2/HER-2 andtumor associated carbohydrate antigens (Cancer, 74 (3):1006-12 (1994)).CTA 16.88, homologous to cytokeratins 8, 18 and 19, is expressed by mostepithelial-derived tumors, including carcinomas of the colon, pancreas,breast, ovary and lung. Thus, antibodies directed to these cytokeratins,such as 16.88 (IgM) and 88BV59 (IgG3k), which recognize differentepitopes on CTA 16.88 (Semin. Nucl. Med., 23 (2):165-79 (1993)), may beemployed as targeting ligands. For targeting colon cancer, anti-CEA IgGFab' fragments may be employed as targeting ligands. Chemicallyconjugated bispecific anti-cell surface antigen, anti-hapten Fab'-Fabantibodies may also be used as targeting ligands. The MG seriesmonoclonal antibodies may be selected for targeting, for example,gastric cancer (Chin. Med. Sci. J., 6 (1):56-59 (1991).

There are a variety of cell surface epitopes on epithelial cells forwhich targeting ligands may be selected. For example, the protein humanpapilloma virus (HPV) has been associated with benign and malignantepithelial proliferations in skin and mucosa. Two HPV oncogenicproteins, E6 and E7, may be targeted as these may be expressed incertain epithelial derived cancers, such as cervical carcinoma. SeeCurr. Opin. Immunol., 6(5):746-54 (1994). Membrane receptors for peptidegrowth factors (PGF-R), which are involved in cancer cell proliferation,may also be selected as tumor antigens. Anticancer Drugs, 5(4):379-93(1994). Also, epidermal growth factor (EGF) and interleukin-2 may betargeted with suitable targeting ligands, including peptides, which bindthese receptors. Certain melanoma associated antigens (MAA), such asepidermal growth factor receptor (EGFR) and adhesion molecules (TumorBiol., 15 (4):188-202 (1994)), which are expressed by malignant melanomacells, can be targeted with the compositions provided herein. The tumorassociated antigen FAB-72 on the surface of carcinoma cells may also beselected as a target.

A wide variety of targeting ligands may be selected for targetingmyocardial cells. Exemplary targeting ligands include, for example,anticardiomyosin antibody, which may comprise polyclonal antibody, Fab'2fragments, or be of human origin, anima origin, for example, mouseorigin, or of chimeric origin. Additional targeting ligands includedipyridamole; digitalis; nifedipine; apolipoprotein; low densitylipoproteins (LDL), including α-LDL, vLDL and methyl LDL; ryanodine;endothelin; complement receptor type 1; IgG Fc; beta 1-adrenergic;dihydropyridine; adenosine; mineralocorticoid; nicotinic acetylcholineand muscarinic acetylcholine; antibodies to the human alpha1A-adrenergic receptor; bioactive agents, such as drugs, including thealpha 1-antagonist prazosin; antibodies to the anti-beta-receptor; drugswhich bind to the anti-beta-receptor; anti-cardiac RyR antibodies;endothelin-1, which is an endothelial cell-derived vasoconstrictorpeptide that exerts a potent positive inotropic effect on cardiac tissue(endothelin-1 binds to cardiac sarcolemmal vesicles); monoclonalantibodies which may be generated to the T-cell receptor α-β receptorand thereby employed to generate targeting ligands; the complementinhibitor sCR1; drugs, peptides or antibodies which are generated to thedihydropyridine receptor; monoclonal antibodies directed towards theanti-interleukin-2 receptor may be used as targeting ligands to directthe present compositions to areas of myocardial tissue which expressthis receptor and which may be up-regulated in conditions ofinflammation; cyclosporine for directing similarly the compositions toareas of inflamed myocardial tissue; methylisobutyl isonitrile; lectinswhich bind to specific sugars on membranes of cardiac myocytes andcardiac endotheli,al cells; adrenomedullin (ADM), which is an endogenoushypotensive and vasorelaxing peptide; atrial natriuretic peptide (ANP);C-type natriuretic peptide (CNP), which is a 22 amino acid peptide ofendothelial cell origin and is structurally related to atrialnatriuretic peptide but genetically distinct, and possesses vasoactiveand antimitogenic activity; vasonatrin peptide (VNP) which is a chimeraof atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP)and comprises 27 amino acids; thrombin; endothelium-derived relaxingfactor (EDRF); neutral endopeptidase 1 (NEP-1); competitive inhibitorsto EDRF, including, for example, NG-monomethyl-L-arginine (L-NMMA);potassium channel antagonists, such as charybdotoxin and glibenclamide;antiheart antibodies, which may be identified in patients withidiopathic dilated cardiomyopathy but which preferably do not elicitcytolysis in the myocardium; antibodies directed against the adeninenucleotide translocator, the branched-chain keto acid dehydrogenase orcardiac myosin; specific antagonists for the endothelin-A receptor,which may be referred to as BQ-123; and antibodies to the angiotensin IIreceptor.

Two of the major antigens of heart sarcolemmal are calcium bindingglycoproteins which copurify with the dihydropyridine receptor. Antiseramay be raised, including polyclonal or monoclonal antibodies, againstpurified sarcolemma. These antibodies may also be employed as targetingligands. Purified fractions of the calcium binding glycoproteins may beisolated from the plasma membranes of the sarcolemma and then used togenerate antibodies. ANP, which, as noted above, may be used as atargeting ligand, can be obtained from cultures of human aorticendothelial cells. ANP is generally localized in endothelium, but alsomay localize to the endothelial or myocardial tissue. ANP may beprepared, for example, using recombinant techniques, as well as bysynthesis of the peptide using peptide synthesis techniques well knownto one skilled in the art. It is also possible to use an antibody,either polyclonal or monoclonal, directed towards ANP. Similarly, apeptide directed to ANP may be used for targeting endothelial and/ormyocardial cells. Both the β and α forms of atrial natriuretic factormay be used as targeting ligands for directing the present compositionsto myocardial tissue.

A wide variety of targeting ligands may be employed to direct thepresent compositions to the GPIIbIIIa receptor. Compositions which aredirected to the GPIIbIIIa receptor are highly useful for targetingvascular thromboses or clots, and are useful for diagnosing and treatingsuch clots. Included among such targeting ligands are, for example,peptides, such as Arg-Gly-Asp-Ser (RGDS), Gly-Arg-Gly-Asp-Ser-Pro(GRGDSP), and Gly-Pro-Arg-Pro (GPRP). Pentapeptides containing thesequence Arg-Gly-Asp (RGD) are also useful including, for example,G4120, which is a cyclic peptide containing the amino acid sequenceArg-Gly-Asp (RGD). Also useful are peptides derived from humancoagulation Factor XIIIA including, for example, fragments such asNKLIVRRGQSFYVQIDFSRPYDPRRDLF RVEYVIGRYPQENKGTYIPVPIVSELQSGKWGAKIVEDRSVRLSIQSS PKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFNPWCEDDAVYLDNEKEREEYVLNDIGVIFYGEVNDIKTRSWSYGQF-R' where R' is--CONH₂ or --NH₂. In addition, peptides which are fragments of theFactor XIIIA fragment, which include in their sequence the sequenceNKLIVRRGOSFYVQIDF SRPYDPRRD or DDAVYLDNEKEREEYVLNDIGVIFYGEVNDIKTRSWSYGQF.

Additional peptides which may be useful as targeting ligands fortargeting the GPIIbIIIa receptor include, for example, peptidescomprising the tripeptide sequence of arginine-tyrosine-aspartic acid(Arg-Tyr-Asp; also abbreviated RGD), linked fromamino-to-carboxy-terminus and which may bind to the GPIIblIIa bindingregion on activated platelets. Exemplary of such peptides include, forexample, peptides of the general formula R¹ -(X¹)_(n)-Arg-Tyr-Asp-(Y)_(o) -(X²)_(m) -R², wherein each of X¹, X² and Y mayindependently be one or more amino acid residues while, in certaincases, it is preferred that Y is other than a serine or alanine residue,and each of m, n and o is independently 0 or 1, provided, in certaincases, that when m is 1, then o is 1, and R¹ is a protected orunprotected terminal amino group and R² is a protected or unprotectedterminal carboxy group. In a preferred embodiment, X¹ is the peptideAla-Arg-Arg-Ser-Ser-Pro-Ser-Tyr-Tyr and X² is the peptideGly-Ala-Gly-Pro-Tyr-Tyr-Ala-Met-Asp-Tyr. Useful peptides includeArg-Ser-Pro-Ser-Tyr-Tyr-Arg-Tyr-Asp-Gly-Ala-Gly-Pro-Tyr-Tyr-Ala-Met-Asp-TyrandAla-Arg-Arg-Ser-Pro-Ser-Tyr-Tyr-Arg-Tyr-Asp-Gly-Ala-Gly-Pro-Tyr-Tyr-Ala-Met-Asp-Tyr.

Synthetic compounds which combine a natural amino acid sequence withsynthetic amino acids can also be used as the targeting ligand, such asa fibrinogen receptor antagonist compound which comprises the sequenceXX-Gly-Asp, wherein XX is a synthetic α-amino acid containing a linearside chain, such as ##STR9## wherein n+n' is 3; AA is a single bond; andR is phenyl or benzyl; or --(CH₂)_(n) --AA--(CH₂)_(n') --NHR wherein nis an integer of 1 to 4; n' is an integer of 2 to 4; AA is oxygen,sulfur or a single bond; and R is H, C₁₋₆ aikyl, optionally substitutedaryl, optionally substituted arylmethyl or optionally substitutedcycloalkyl, provided, in certain cases, that when AA is a single bondand R is H, then n+n' is other than 3 or 4.

Another such compound comprises a fibrinogen receptor antagonist of theformula: ##STR10## wherein XX is a synthetic α-amino acid containing alinear side chain having the formula ##STR11## wherein n+n' is 3; AA isa single bond; and R is phenyl or benzyl; or --(CH₂)_(n)--AA--(CH₂)_(n') --NHR, wherein n is an integer of 1 to 4; n' is aninteger of 2 to 4; AA is oxygen, sulfur or a single bond; and R is H,C₁₋₆ alkyl, optionally substituted cycloalkyl, provided that, in certaincases, when AA is a single bond and R is H, then n+n' is other than 3 or4, and ZZ is a sequence of 1 to 4 optionally substituted amino acids.

Other useful peptides for use as targeting ligands include, for example,Elegantin, which has the following sequence:Gly-Glu-Glu-Cys-Asp-Cys-Gly-Ser-Pro-Glu-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu-Arg-Pro-Gly-Ala-Gln-Cys-Ala-Asp-Gly-Leu-Cys-Cys-Asp-Gln-Cys-Arg-Phe-Lys-R-R'-Arg-Thr-Ile-Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asn-Pro-Asp-Asp-Arg-Cys-Thr-Gly-Gln-Ser-Ala-Asp-Cys-Pro-Arg-Asn-Gly-Tyr,wherein each of R and R' is independently any amino acid; Albolabrin,which has the following sequence:Glu-Ala-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu-Leu-Pro-Gly-Ala-Gln-Cys-Gly-Glu-Gly-Leu-Cys-Cys-Asp-Gln-Cys-Ser-Phe-Met-Lys-Lys-Gly-Thr-Ile-Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Ile-Ser-Ala-Gly-Cys-Pro-Arg-Asn-Pro-Leu-His-Ala-Batroxostatin, which has the following sequence:Glu-Ala-Gly-Glu-Glu-Cys-Asp-Cys-Gly-Thr-Pro-Glu-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu-Arg-Pro-Gly-Ala-Gln-Cys-Ala-Glu-Gly-Leu-Cys-Cys-Asp-Gln-Cys-Arg-Phe-Lys-Gly-Ala-Gly-Lys-Ile-Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asn-Pro-Asp-Asp-Cys-Thr-Gly-Gln-Ser-Ala-Asp-Cys-Pro-Arg-Phe;and Flavoridin, which has the following sequence:Gly-Gly-Glu-Cys-Asp-Cys-Gly-Ser-Pro-Glu-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu-Arg-Pro-Gly-Ala-Gln-Cys-Ala-Asp-Gly-Leu-Cys-Cys-Asp-Gln-Cys-Arg-Phe-Lys-R-R'-Arg-Thr-Ile-Cys-Arg-Ile-Ala-Arg-Gly-Asp-Phe-Pro-Asp-Asp-Arg-Cys-Thr-Gly-Leu-Ser-Ala-Asp-Cys-Pro-Arg-R-Asn-Asp-Leu,wherein each of R and R' is independently any amino acid.

Other ligands useful for targeting the GPIIbIIIa receptor includesynthetic compounds, such as Ac-(D)Phe-Pro-boroArg and the cyclicpeptidomimeticcyclo(D-2-aminobutyrate-N-Methyl-L-Arginyl-Glycyl-L-Aspartyl-3-amino-methyl-benzoicacid) methanesulfonate salt. Peptides that can also be used include alibrary of hexapeptides flanked by cysteine residues (capable of formingcyclic disulfides) and cyclic, disulfide-bonded forms of peptides withthe sequence Arg-Gly-Asp or Lys-Gly-Asp, as well as thecarboxyl-terminal derived peptide, REYVVMWK. Certain matrixglycoproteins such as Thrombospondin are also useful in this regard.Members of the serpin family of serine protease inhibitors, such asPlasminogen activator inhibitor type 1 (PAI-1) are other useful ligands.

Generally, it is preferred to employ, as targeting ligands for theGPIIbIIIa receptor, a peptide having from about 3 to about 20 aminoacids, with peptides having from about 4 to about 15 amino acids beingmore preferred. Even more preferably, targeting ligands for theGPIIbIIIa receptor may comprise peptides having from about 4 to about 8amino acids, with peptides having from about 4 to about 6 amino acids orabout 5 amino acids being still more preferred. If desired, the peptidesmay be cyclized, for example, by (1) side chain-to-side chain covalentlinkages, including, for example, by the formation of a disulfidelinkage via the oxidation of two thiol containing amino acids cr analogsthereof, including, for example, cysteine or penicillamine; (2)end-to-side chain covalent linkages, including, for example, by the useof the amino terminus of the amino acid sequence and a side chaincarboxylate group, such as, for example, a non-critical glutamic acid oraspartic acid group. Alternatively, the end-to-side chain covalentlinkage may involve the carboxylate terminus of the amino acid sequenceand a side chain amino, amidine, guanidine, or other group in the sidechain which contains a nucleophilic nitrogen atom, such side chaingroups including, for example, lysine, arginine, homoarginine,homolysine, or the like; (3) end-to-end covalent linkages that arecovalent amide linkages, or the like. Such processes are well known toone skilled in the art. In addition, "pseudo-cyclization" may beemployed, in which cyclization occurs via non-covalent interactions,such as electrostatic interactions, which induces a folding of thesecondary structure to form a type of cyclic moiety. Metal ions may aidthe induction of a "pseudocyclic" formation. This type of pseudocyclicformation may be analogous to "zinc fingers." As known to one ofordinary skill in the art, zinc fingers involve the formation due toelectrostatic interactions between a zinc ion (Zn²⁺) and cysteine,penicillamine and/or homocysteine, of a region in the shape of a loop(the finger). In the case of homocysteine, the RGD sequence would resideat the tip of the finger. Of course, it is recognized that, in thecontext of the present invention, any type of stabilizing cyclizationwould be suitable as long the recognition and binding peptide ligand,such as, for example, RGD, maintains the proper conformation and/ortopography to bind to the appropriate receptor in clots with areasonable Michaelis-Menten constant (k_(m)) or binding constant. Asused herein, the term "conformation" refers to the three-dimensionalorganization of the backbone of the peptide, peptoid, or pseudopeptide,and the term "topography" refers to the three-dimensional organizationof the sidechain of the peptide, peptoid, or pseudopeptide.

Other suitable targeting ligands include the following compounds:Ac-Cys-Arg-Gly-Asp-Met-Phe-Gly-Cys-CONH₂ ;Ac-Cys-Arg-Gly-Asp-Met-Leu-Arg-Cys-CONH₂ ;Ac-Cys-Arg-Gly-Asp-Phe-Leu-Asn-Cys-CONH₂ ;Ac-Cys-Asn-Thr-Leu-Lys-Gly-Asp- Cys-CONH₂ ;Ac-Cys-Asn-Trp-Lys-Arg-Gly-Asp-Cys-CONH₂ ; andAc-Cys-N-methyl-Arg-Gly-Asp-Pen-CONH₂, where "Pen" refers topenicillamine (β,β-dimethylcysteine).

Other compounds which may be used as targeting ligands include peptides,or derivatives thereof, represented by the formula

    A-B-Arg-Gly-Asp-C-D

wherein A is proline, thioproline, hydroxyproline, dehydroproline,2-oxo-4-thiazolidine carboxylic acid, N-alkyl glycine or an amino acidderivative of the formula ##STR12## tryptophan, or a tryptophanderivative of the formula ##STR13## pyroglutamic acid or2-azetidinone-4-carboxylic acid B is serine, glycine, valine, alanine,threonine or β-alanine; C is an amino acid group having a hydrophobicfunctional group; and D is hydroxy or amino; wherein R₁ is hydrogen,--(CH₂)_(p) CH₃ or --CO--(CH₂)_(p) CH₃ ; R₂ is hydrogen or alkyl; R₃ ishydrogen or alkoxy; R₄ is hydrogen or alky; R₅ is hydrogen, amino oracylamino; m is an integer of 2 to 5; n is an integer of 0 to 2; p is aninteger of 0 to 5; and q is an integer of 0 to 3.

Another targeting ligand which may be suitable for use in connectionwith the present compositions is a peptide, a peptide derivative, or asalt thereof having the formula

    A-B-Arg-Gly-Asp-C-D

where A is arotic acid or hydroorotic acid; B is an amino acid; C is anamino acid having a hydrophobic functional group; and D is hydroxy oramino. In the above compounds, examples of amino acids havinghydrophobic functional groups in the definition of "C" are tryptophanand phenylalanine.

Various peptides which would be suitable for use as a targeting ligandin the present invention, especially for targeting GPIIbIIIa, aredescribed, for example, in U.S. Pat. No. 5,498,601 and European PatentApplications: 0 368 486 A2, 0 382 451 A2, and 0 422 938 B1, thedisclosures of which are hereby incorporated herein by reference intheir entirety. Other targeting ligands which may be used in thecompositions of the present invention, in addition to those exemplifiedabove, would be apparent to one of ordinary skill in the art in view ofthe present disclosure. Other suitable targeting ligands include, forexample, conjugated peptides, such as, for example, glycoconjugates andlectins, which are peptides attached to sugar moieties. The compositionsmay comprise a single targeting ligand, as well as two or more differenttargeting ligands.

The targeting ligand is preferably covalently bound to the surface ofthe composition by a spacer including, for example, hydrophilicpolymers, preferably polyethylene glycol. Preferred molecular weights ofthe polymers are from 1000 da to 10,000 da, with 500 da being mostpreferred. Preferably the polymer is bifunctional with the targetingligand bound to a terminus of the polymer. Generally, the targetingligand will range from about 0.1 to about 20 mole % of the exteriorcomponents of the vesicle. In the case of gas-filled lipid vesicles,this amount is preferably between about 0.5 and about 10 mole % withabout 1 to about 10 mole % most preferred. The exact ratio will dependupon the particular targeting ligand.

In another embodiment, the targeting ligands are directed to lymphocyteswhich may be T-cells or B-cells, with T-cells being the preferredtarget. Depending on the targeting ligand, the composition may betargeted to one or more classes or clones of T-cells. To select a classof targeted lymphocytes, a targeting ligand having specific affinity forthat class is employed. For example, an anti CD-4 antibody can be usedfor selecting the class of T-cells harboring CD-4 receptors, an antiCD-8 antibody can be used for selecting the class of T-cells harboringCD-8 receptors, an anti CD-34 antibody can be used for selecting theclass of T-cells harboring CD-34 receptors, etc. A lower molecularweight ligand is preferably employed, e.g., Fab or a peptide fragment.For example, an OKT3 antibody or OKT3 antibody fragment may be used.When a receptor for a class of T-cells or clones of T-cells is selected,the composition will be delivered to that class of cells. UsingHLA-derived peptides, for example, will allow selection of targetedclones of cells expressing reactivity to HLA proteins.

The ultimate purpose of the linkage between the targeting ligand and thetarget may be the delivery of the composition to the cell forendocytosis or fusion. Although not intending to be bound by anyparticular theory of operation, once the composition has linked to itstarget, the composition may gain access to the interior of the targetcell either through a fusion-initiated capping and patching mechanism,the intervention of clathrin-coated pits or through classicalendocytosis, depending on the mechanisms for engulfment peculiar to thetarget cell, or by other natural or induced means. One skilled in theart will recognize the potential for targeted uses of bioactive agentswhich gain access to the target cells or tissue via ligand-receptorbinding.

The following tables illustrate ligands from the majorhistocompatability complex (MHC) and their receptors in the class ofT-cells for which they have affinity. All the ligands, T-cell receptorsand peptide sequences in the table below may be used in the presentinvention.

                  TABLE 2                                                         ______________________________________                                        MHC LIGANDS AND T-CELL RECEPTORS                                              T-Cell Receptor                                                                          Ligand       Peptide Sequence                                      ______________________________________                                        HTB157.7   K.sup.b (Q10b hybrid)                                                                      Heterogeneous                                         HTB157.7   pK.sup.b 163-174                                                                           NA                                                    2C         L.sup.d /p2Ca                                                                              LSPFPFDL*                                             2C         L.sup.d /p2Ca-A5                                                                           LSPFAFDL                                              2C         L.sup.d /p2Ca-A3                                                                           LSAFPFDL                                              2C         L.sup.d /p2Ca-A8                                                                           LSPFPFDA                                              2C         L.sup.d /SL9 SPFPFDLLL                                             2C         K.sup.b /p2Ca                                                                              LSPFPFDL                                              2C         L.sup.d /QL9 QLSPSPDL                                              4G3        K.sup.b /pOV8                                                                              SIINFEKL                                              2C         L.sup.d /p2Ca-Y4                                                                           LSPYPFDL                                              2C         L.sup.d /p2Ca-A1                                                                           ASPFPFDL                                              Clone 30   K.sup.b /lgG (bivalent)                                                                    Heterogeneous                                         14.3d      1-E.sup.d /pHA                                                                             SSFGAFGIFPK                                           5C.C7      1-E.sup.k /MCC                                                                             ANERADLIAYLKQATK                                      228.4      1-E.sup.k /MCC-K99A                                                                        ANERADLIAYLKQATK                                      2B4        1-E.sup.k /MCC                                                                             ANERADLIAYLKQATK                                      2B4        1-E.sup.k /PCC                                                                             ANERADLIAYLKQATAK                                     2B4        1-E.sup.k /MCC-T102S                                                                       ANERADLIAYLKQASK                                      HA1.7      SEB                                                                14.3d β                                                                             SEC1                                                               14.3d β                                                                             SEC2                                                               14.3d β                                                                             SEC3                                                               14.3d β                                                                             SEB                                                                14.3d β                                                                             SPEA                                                               ______________________________________                                         *Single-letter code for amino acids. Summarized from Fremont et al,           Current Opinion In Immunology, (1996) 8:93100, page 96, Table 2, the          disclosure of which is hereby incorporated herein by reference in its         entirety.                                                                

Another major area for targeted delivery involves the interlekin-2(IL-2) system. L-2 is a t-cell growth factor produced following antigenor mitogen induced stimulation of lymphoid cells. Among the cell typeswhich produce IL-2 are CD4⁺ and CD8⁺ t-cells and large granularlymphocytes, as well as certain t-cell tumors. IL-2 receptors areglycoproteins expressed on responsive cells. They are notable inconnection with the present invention because they are readilyendocytosed into lysosomal inclusions when bound to IL-2. The ultimateeffect of this endocytosis depends on the target cell, but among thenotable in vivo effects are regression of transplantable murine tumors,human melanoma or renal cell cancer. IL-2 has also been implicated inantibacterial and antiviral therapies and plays a role in allograftrejection. In addition to IL-2 receptors, preferred targets include theanti-IL-2 receptor antibody, natural IL-2 and an IL-2 fragment of a20-mer peptide or smaller generated by phage display which binds to theIL-2 receptor.

Although not intending to be bound by any particular theory ofoperation, IL-2 can be conjugated to the compositions and thus mediatethe targeting of cells bearing IL-2 receptors. Endocytosis of theligand-receptor complex would then deliver the composition to thetargeted cell, thereby inducing its death through apoptosis--independentand superceding any proliferative or activiating effect which IL-2 wouldpromote alone.

Additionally, an IL-2 peptide fragment which has binding affinity forIL-2 receptors can be incorporated either by direct attachment to areactive moiety on the composition or via a spacer or linker moleculewith a reactive end such as an amine, hydroxyl, or carboxylic acidfunctional group. Such linkers are well known in the art and maycomprise from 3 to 20 amino acid residues. Alternatively, D-amino acidsor derivatized amino acids may be used which avoid proteolysis in thetarget tissue.

Still other systems which can be used in the present invention includeIgM-mediated endocytosis in B-cells or a variant of the ligand-receptorinteractions described above wherein the T-cell receptor is CD2 and theligand is lymphocyte function-associated antigen 3 (LFA-3), asdescribed, for example, by Wallner et al, J. Experimental Med.,166:923-932 (1987), the disclosure of which is hereby incorporated byreference herein in its entirety.

The targeting ligand may be incorporated in the present compositions ina variety of ways. Generally speaking, the targeting ligand may beincorporated in the present compositions by being associated covalentlyor non-covalently with one or more of the lipids which are included inthe compositions.

Exemplary covalent bonds by which the targeting ligands are associatedwith the compositions include, for example, amide (--CONH--); thioamide(--CSNH--); ether (ROR'), where R and R' may be the same or differentand are other than hydrogen); ester (--COO--); thioester (--COS--);--O--; --S--; --S_(n) --, where n is greater than 1, preferably about 2to about 8, and more preferably about 2; carbamates; --NH--; --NR--,where R is alkyl, for example, alkyl of from 1 to about 4 carbons;urethane; and substituted imidate; and combinations of two or more ofthese. Covalent bonds between targeting ligands and lipids may beachieved through the use of molecules that may act as spacers toincrease the conformational and topographical flexibility of the ligand.Examples of such spacers include, for example, succinic acid,1,6-hexanedioic acid, 1,8-octanedioic acid, and the like, as well asmodified amino acids, such as, for example, 6-aminohexanoic acid,4-aminobutanoic acid, and the like. In addition, in the case oftargeting ligands which comprise peptide moieties, side chain-to-sidechain crosslinking may be complemented with side chain-to-endcrosslinking and/or end-to-end crosslinking. Also, small spacermolecules, such as dimethylsuberimidate, may be used to accomplishsimilar objectives. The use of agents, including those used in Schiffsbase-type reactions, such as gluteraldehyde, may also be employed. TheSchiffs base linkages, which may be reversible linkages, can be renderedmore permanent covalent linkages via the use of reductive aminationprocedures. This may involve, for example, chemical reducing agents,such as lithium aluminum hydride reducing agents or their milderanalogs, including lithium aluminum diisobutyl hydride (DIBAL), sodiumborohydride (NaBH₄) or sodium cyanoborohydride (NaBH₃ CN).

The covalent linking of the targeting ligands to the presentcompositions may be accomplished using synthetic organic techniqueswhich would be readily apparent to one of ordinary skill in the art inview of the present disclosure. For example, the targeting ligands maybe linked to the materials, including the lipids, via the use of wellknown coupling or activation agents. As known to the skilled artisan,activating agents are generally electrophilic, which can be employed toelicit the formation of a covalent bond. Exemplary activating agentswhich may be used include, for example, carbonyldiimidazole (CDI),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), methylsulfonyl chloride, Castro's Reagent, and diphenyl phosphoryl chloride.

The covalent bonds may involve crosslinking and/or polymerization.Cross-linking preferably refers to the attachment of two chains ofpolymer molecules by bridges, composed of either an element, a group, ora compound, which join certain carbon atoms of the chains by covalentchemical bonds. For example, crosslinking may occur in polypeptideswhich are joined by the disulfide bonds of the cystine residue.Crosslinking may be achieved, for example, by (1) adding a chemicalsubstance (crosslinking agent) and exposing the mixture to heat, or (2)subjecting a polymer to high energy radiation. A variety of crosslinkingagents, or "tethers", of different lengths and/or functionalities aredescribed, for example, by Lunbland, Techniques in Protein Modification,CRC Press, Inc., Ann Arbor, Mich., pp. 249-68 (1995), the disclosures ofwhich is hereby incorporated herein by reference in its entirety.Exemplary crosslinkers include, for example,3,3'-dithiobis(succinimidylpropionate), dimethyl suberimidate, and itsvariations thereof, based on hydrocarbon length, andbis-N-maleimido-1,8-octane.

The targeting ligands may be linked or attached to the compositions ofthe present invention via a linking group. A variety of linking groupsare available and would be apparent to one skilled in the art in view ofthe present disclosure. Preferably, the linking group comprises ahydrophilic polymer. Suitable hydrophilic polymers include, for example,polyalkyleneoxides such as, for example, polyethylene glycol (PEG) andpolypropylene glycol (PPG), polyvinylpyrrolidones,polyvinylmethylethers, polyacrylamides, such as, for example,polymethacrylamides, polydimethylacrylamides andpolyhydroxypropylmethacrylamides, polyhydroxyethyl acrylates,polyhydroxypropyl methacrylates, polyalkyloxazolines, such aspolymethyloxazolines and polyethyloxazolines,polyhydroxyalkyloxazolines, such as polyhydroxyethyloxazolines,polyhyhydroxypropyloxazolines, polyvinyl alcohols, polyphosphazenes,poly(hydroxyalkylcarboxylic acids), polyoxazolidines, polyaspartamide,and polymers of sialic acid (polysialics). The hydrophilic polymers arepreferably selected from the group consisting of PEG, PPG,polyvinylalcohol and polyvinylpyrrolidone and copolymers thereof, withPEG and PPG polymers being more preferred and PEG polymers being evenmore prefered. Thus, in embodiments involving lipid compositions whichcomprise lipids bearing polymers including, for example, DPPE-PEG, thetargeting ligand may be linked directly to the polymer which is attachedto the lipid to provide, for example, a conjugate of DPPE-PEG-TL, whereTL is a targeting ligand. Thus, using the example DPPE-PEG, such as, forexample, DPPE-PEG5000, the aforementioned conjugate may be representedas DPPE-PEG5000-TL. The hydrophilic polymer used as a linking group ispreferably a bifunctional polymer, for example, bifunctional PEG, suchas diamino-PEG. In this case, one end of the PEG group is linked, forexample, to a lipid compound, and is bound at the free end to thetargeting ligand via an amide linkage. A hydrophilic polymer, forexample, PEG, substituted with a terminal carboxylate group on one endand a terminal amino group on the other end, may also be used. Theselatter bifunctional hydrophilic polymer may be preferred since theypossess various similarities to amino acids.

Standard peptide methodology may be used to link the targeting ligand tothe lipid when utilizing linker groups having two unique terminalfunctional groups. Bifunctional hydrophilic polymers, and especiallybifunctional PEGs, may be synthesized using standard organic syntheticmethodologies. In addition, many of these materials are availablecommercially, such as, for example, α-amino-ω-carboxy-PEG which iscommercially available from Shearwater Polymers (Huntsville, Ala.). Anadvantage of using a PEG material as the linking group is that the sizeof the PEG can be varied such that the number of monomeric subunits ofethylene glycol may be as few as, for example, about 5, or as many as,for example, about 500 or more. Accordingly, the "tether" or length ofthe linkage may be varied, as desired. This may be important depending,for example, on the particular targeting ligand employed. For example, atargeting ligand which comprises a large protein molecule may require ashort tether, such that it will simulate a membrane bound protein. Ashort tether would also allow for a vesicle to maintain a closeproximity to the cell. This can be used advantageously in connectionwith vesicles which also comprise a bioactive agent in that theconcentration of bioactive agent which is delivered to the cell may beadvantageously increased.

Another suitable linking group which may provide a short tether isglyceraldehyde. Glyceraldehyde may be bound, for example, to DPPE via aSchiff's base reaction. Subsequent Amadori rearrangement can provide asubstantially short linking group. The β carbonyl of the Schiff's basemay then react with a lysine or arginine of the targeting protein orpeptide to form the targeted lipid.

More specifically, the compositions of the present invention may containvarious functional groups, such as, for example, hydroxy, thio and aminegroups, which can react with a carboxylic acid or carboxylic acidderivative of the hydrophilic polymeric linker using suitable couplingconditions which would be apparent to one of ordinary skill in the artin view of the present disclosure. After the carboxylic acid group (orderivative thereof) reacts with the functional group, for example,hydroxy, thio or amine group to form an ester, thioester or amide group,any protected functional group may be deprotected utilizing procedureswhich would be well known to one skilled in the art. The term protectinggroup refers to any moiety which may be used to block the reaction of afunctional group and which may be removed, as desired, to afford theunprotected functional group. Any of a variety of protecting groups maybe employed and these will vary depending, for example, as to whetherthe group to be protected is an amine, hydroxyl or carboxyl moiety. Ifthe functional group is a hydroxyl group, suitable protecting groupsinclude, for example, certain ethers, esters and carbonates. Suchprotecting groups are described, for example, in Greene, TW and Wuts,PGM "Protective Groups in Organic Synthesis" John Wiley, New York, 2ndEdition (1991), the disclosure of which is hereby incorporated herein byreference in its entirety. Protecting groups for amine groups include,for example, t-butyloxycarbony (Boc), allyloxycarbonyl (Alloc),benzyloxycarbonyl(Cbz), o-nitrobenzyloxycarbonyl and andtrifluoroacetate (TFA).

Amine groups which may be present, for example, on a backbone of apolymer which is included in the vesicles, may be coupled to aminegroups on a hydrophilic polymer by forming a Schiff s base, for example,by using coupling agents, such as glutaraldehyde. An example of thiscoupling is described by Allcock et al, Macromolecules, 19(6):1502-1508(1986), the disclosure of which is hereby incorporated herein byreference in its entirety. If, for example, vesicles are formulated frompolylysine, free amino groups may be exposed on the surface of thevesicles, and these free amine groups may be activated as describedabove. The activated amine groups can be used, in turn, to couple to afunctionalized hydrophilic polymer, such as, for example,α-amino-ω-hydroxy-PEG in which the ω-hydroxy group has been protectedwith a carbonate group. After the reaction is completed, the carbonategroup can be cleaved, thereby enabling the terminal hydroxy group to beactivated for reaction to a suitable targeting ligand. In certainembodiments, the surface of a vesicle may be activated, for example, bydisplacing chlorine atoms in chlorine-containing phosphazene residues,such as polydichlorophosphazene. Subsequent addition of a targetingligand and quenching of the remaining chloride groups with water oraqueous methanol will yield the coupled product.

In addition, poly(diphenoxyphosphazene) can be synthesized (Allcock etal., Macromolecules, 19(6):1502-1508 (1986)) and immobilized, forexample, on DPPE, followed by nitration of the phenoxy moieties by theaddition of a mixture of nitric acid and acetic anhydride. Thesubsequent nitro groups may then be activated, for example, by (1)treatment with cyanogen bromide in 0.1 M phosphate buffer (pH 11),followed by addition of a targeting ligand containing a free aminomoiety to generate a coupled urea analog, (2) formation of a diazoniumsalt using sodium nitrite/HCl, followed by addition of the targetingligand to form a coupled ligand, and/or (3) the use of a dialdehyde, forexample, glutaraldehyde as described above, to form a Schiff's base.After linking the DPPE to the hydrophilic polymer and the targetingligand, the vesicles may be formulated utilizing the proceduresdescribed herein.

Aldehyde groups on polymers can be coupled with amines as describedabove by forming a Schiff's base. An example of this coupling procedureis described in Allcock and Austin, Macromolecules, 14:1616 (1981), thedisclosure of which is hereby incorporated herein by reference in itsentirety.

In the above procedures, the polymer or terminus of the lipid, forexample, phosphatidylglycerol or phosphatidylethanolamine, is preferablyactivated and coupled to the hydrophilic polymeric linker, the terminusof which has been blocked in a suitable manner. As an example of thisstrategy, α-amino-ω-carboxy-PEG4000 having a t-Boc protected terminalamino group and a free carboxylate end, may be activated with1,1'-carbonyl-diimidazole in the presence of hydroxybenzotriazole inN-methylpyrollidone. After the addition of phosphatidylethanolamine, thet-Boc group may be removed by using trifluoro-acetic acid (TFA), leavingthe free amine. The amine may then be reacted with a targeting ligandwhich may comprise, for example, a peptide, protein, alkaloid, or othermoiety, by similar activation of the ligand, to provide thelipid-linker-targeting ligand conjugate. Other strategies, in additionto those exemplified above, may be utilized to prepare thelipid-linker-targeting ligand conjugates. Generally speaking, thesemethods employ synthetic strategies which are generally known to oneskilled in the art of synthetic organic chemistry.

As known to one of ordinary skill in the art, immunoglobulins typicallycomprise a flexible region which is identified as the "hinge" region.See, e.g., "Concise Encyclopedia of Biochemistry", Second Edition,Walter de Gruyter & Co., pp. 282-283 (1988). Fab' fragments can belinked to the present compositions using the well-defined sites of thethiols of the hinge region. This is a preferred region for coupling Fab'fragments as the potential binding site is remote from theantigen-recognition site. Generally, it may be difficult to utilize thethiols of the hinge group unless they are adequately prepared. Inparticular, as outlined by Shahinian and Salvias (Biochimica etBiophysica Acta, 1239:157-167 (1995)) it may be important to reduce thethiol group so that they are available for coupling, for example, tomaleimide derivatized linking groups. Examples of reducing agentscommonly used are ethanedithiol, mercaptoethanol, mercaptoethylamine orthe more commonly used dithiothreitol, commonly referred to as Cleland'sreagent. However, it should be noted that care should be exercised whenutilizing certain reducing agents, such as dithiothreitol, asover-reduction may result. Discriminating use of reducing agents may benecessary in connection with proteins whose activity or binding capacitymay be compromised due to overreduction and subsequent denaturation orconformational change. See, Shahinian et al, Biochim. Biophys. Acta,1239:157-167 (1995), the disclosure of which is hereby incorporatedherein by reference in its entirety.

F(ab')₂ antibody fragments may be prepared by incubating the antibodieswith pepsin (60 μg/ml) in 0.1 M sodium acetate (pH 4.2) for 4 h at 37°C. Digestion may be terminated by adding 2 M Tris (pH 8.8) to a finalconcentration of 80 mM. The F(ab')₂ fragments may then be obtained bycentrifugation (10,000×g. 30 min. 4° C.). The supernatant may then bedialyzed at 4° C. against 150 mM NaCl, 20 mM phosphate at pH 7.0. Thisthen may be chromatographed on a column of Protein A-Sepharose CL-4B toremove any undigested IgG. The Fab' fragments may then be prepared byextensively degassing the solutions and purging with nitrogen prior touse. The F(ab')₂ fragments may be provided at a concentration of 5 mg/mland reduced under argon ink 30 mM cysteine. Alternatively, cysteaminemay be employed. 100 mM Tris, pH 7.6 may be used as a buffer for 15 minat 37° C. The solutions may then be diluted 2-fold with an equal volumeof the appropriate experimental buffer and spun through a 0.4 ml spincolumn of Bio-Gel P-6DG. The resulting Fab' fragments may be moreefficient in their coupling to maleimide linkers. Note also that thesame procedure may be employed with other macromolecules containingcysteine residues for coupling, for example, to the maleimide spacers.Also, peptides may be utilized provided that they contain a cysteineresidue. If the peptides have not been made fresh and there is apossibility of oxidation of cysteine residues within the peptidestructure, it may be necessary to regenerate the thiol group using theapproach outlined above.

Additional linkers would include other derivatives of lipids useful forcoupling to a bifunctional spacer. For example, phosphatidylethanolamine(PE) may be coupled to a bifunctional agent. For example N-succinimidyl4-(p-maleimidophenyl)-butyrate (SMPB) and N-succinimidyl3-(2-pyridyldithiol) propionate (SPDP), N-succinimidyltrans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate (SMCC), andN-succinimidyl 3-maleimidyl-benzoate (SMB) may be used among others, toproduce, for example the functionalized lipids MPB-PE and PDP-PE.

The free end of the hydrophilic spacer, such as polyethylene glycolethylamine, which contains a reactive group, such as an amine orhydroxyl group, could be used to bind a cofactor or other targetingligand. For example, polyethylene glycol ethylamine may be reacted withN-succinimidylbiotin or p-nitrophenylbiotin to introduce onto the spacera useful coupling group. For example, biotin may be coupled to thespacer and this will readily bind non-covalently proteins. As anexample, MPB-PEG-DPPE may be synthesized as follows. DPPE-PEG with afree amino group at the terminus of the PEG will be provided asdescribed previously. Synthesis of the SMPB:PEG-DPPE may then be carriedout with 1 equivalent of triethylamine in chloroform at a molar ratio of1:5 SMPB:DPPE-PEG. After 3 hours, the reaction mixture will beevaporated to dryness under argon. Excess unreacted SMPB and major byproducts will be removed by preparative thin layer chromatography (TLC,silica gel developed with 50% acetone in chloroform). The upper portionof the lipid band can be extracted from the silica with about 20-30%methanol in chloroform (V:V) resulting in the isolation of pure intactMPB-Peg-DPPE. Streptavidin may then be coupled to proteins so that theproteins in turn may then be coupled to the MPB-PEG-DPPE. Briefly SPDPwould be incubated with streptavidin at room temperature for 30 minutesand chromatography employed to remove unreacted SPDP. Dithiothreitol(DTT) was added to the reaction mixture and 10 minutes later2-thiopyridone at a concentration of 343 nM. The remainder of thereaction mixture is reduced with DTT (25 mM for 10 min.). The thiolatedproduct is isolated by gel exclusion. The resulting streptavidin labeledproteins may then be used to bind to the biotinylated spacers affixed tothe lipid moieties.

In preferred embodiments, the targeted compositions may be used to formtargeted emulsions and/or targeted vesicles, including, for example,targeted cochleates, targeted emulsions, targeted micelles, and/ortargeted liposomes. The targeting ligand which is attached to thecompositions from which the vesicles are prepared may be directed, forexample, outwardly from the surface of the vesicle. Thus, there isprovided a targeted vesicle which can be used to target receptors and.tissues.

In certain embodiments, the targeting ligands may be incorporated in thepresent compositions via non-covalent associations. As known to oneskilled in the art, non-covalent association is generally a function ofa variety of factors, including, for example, the polarity of theinvolved molecules, the charge (positive or negative), if any, of theinvolved molecules, the extent of hydrogen bonding through the molecularnetwork, and the like. Non-covalent interactions may be employed to bindthe targeting ligand to the lipid, or directly to the surface of avesicle. For example, the amino acid sequence Gly-Gly-His may be boundto the surface of a vesicle, preferably by a linker, such as PEG, andcopper, iron or vanadyl ion may then be added. Proteins, such asantibodies which contain histidine residues, may then bind to thevesicle via an ionic bridge with the copper ion, as described in U.S.Pat. No. 5,466,467, the disclosure of which is hereby incorporatedherein by reference in its entirety. An example of hydrogen bondinginvolves cardiolipin lipids which can be incorporated into the lipidcompositions.

In preferred embodiments of the present invention, which may involvevesicles, changes, for example, in pH and/or temperature in vivo, may beemployed to promote a change in location in the targeting ligands, forexample, from a location within the vesicle, to a location external tothe outer wall of the vesicle. This may promote binding of the targetingligands to targeting sites, for example, receptors, such as lymphocytes,and tissues, including myocardial, endothelial and epithelial cells,since the targeting ligand has a greater likelihood of exposure to suchtargeting sites. In addition, high energy ultrasound can be used topromote rupturing of the vesicles. This can also expose the targetingligand to the desired binding site.

As an example, a targeting ligand incorporated into the compositions ofthe present invention may be of the formula:

    L-P-T

wherein L is a lipid, surfactant, bioactive agent or the like; P is ahydrophilic polymer; and T is a targeting ligand.

In a preferred embodiment, L is a lipid selected from the groupconsisting of lecithins, phosphatidylcholines, phosphatidylserines,phosphatidylinositols, cardiolipins, cholesterols, cholesterolamines,lysophosphatides, erythrosphingosines, sphingomyelins, ceramides,cerebrosides, saturated phospholipids, unsaturated phospholipids, andkrill phospholipids. More preferably, L is a lipid is selected from thegroup consisting of lecithins, phosphatidylcholines, phosphatidylserinesand phosphatidylinositols. In other preferred embodiments, L is a lipidselected from the group consisting of1,2-diacyl-sn-glycero-3-phosphocholines, 1,2-diacyl-sn-glycero-3-phosphoethanolamines,1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerols)],1,2-diacyl-sn-glycero-3-phosphates,1,2-diacyl-sn-glycero-3-[phosphoserines], lysophosphatidyl-cholines,lysophosphatidylglycerols, 1,2-diacyl-sn-glycerols, 1,2-diacyl-ethyleneglycols,N-(n-caproylamine)-1,2-diacyl-sn-glycero-3-phosphoethanolamines,N-dodecanylamine-1,2-diacyl-sn-glycero-3-phosphoethanol- amines,N-succinyl-1,2-diacyl-sn-glycero-3-phosphoethanolamines, N-glutaryl-1,2-diacyl-sn-glycero-3 -phosphoethanolamines andN-dodecanyl-1,2-diacyl-sn-glycero-3-phosphoethanol-amines. Morepreferably, L is a lipid selected from the group consisting of1,2-diacyl-sn-glycero-3-phosphocholines,1,2-diacyl-sn-glycero-3-phosphoethanolamines,1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerols)],1,2-diacyl-sn-glycero-3-phosphates,1,2-diacyl-sn-glycero-3-[phosphoserines], lysophosphatidylcholines,lysophosphatidyl-glycerols and 1,2-diacyl-sn-glycerols.

In other preferred embodiments, L is a surfactant, preferably afluorosurfactant, and more preferably a fluorosurfactant havingpolyethylene glycol attached thereto.

In the above compounds, P is a hydrophilic polymer. Preferably, P is ahydrophilic polymer selected from the group consisting ofpolyalkyleneoxides, polyvinyl alcohol, polyvinylpyrrolidones,polyacrylamides, polymethacrylamides, polyphosphazenes, phosphazene,poly(hydroxyalkylcarboxylic acids) and polyoxazolidines. Morepreferably, P is a polyalkyleneoxide polymer, with polyethylene glycoland polypropylene glycol being even more preferred and polyethyleneglycol being particularly preferred.

In the above formula, T is a targeting ligand. Preferably, T is atargeting ligand selected from the group consisting of proteins,peptides, saccharides, steroids, steroid analogs, and genetic material,with proteins, peptides and saccharides being more preferred.

In the case of targeting ligands which comprise saccharide groups,suitable saccharide moieties include, for example, monosaccharides,disaccharides and polysaccharides. Exemplary monosaccharides may havesix carbon atoms and these saccharides include allose, altrose, glucose,dextrose, mannose, gulose, idose, galactose, talose, fructose, psicose,verbose and tagatose. Five carbon saccharides include ribose, arabinose,xylose, lyxose, ribulose and xylulose. Four carbon saccharides includeerythrose, threose and erythrulose. Disaccharides include sucrose,lactose, maltose, isomaltose and cellobiose. Saccharide bearingtargeting lipids may be, synthesized through a multistep organicsynthesis approach, as described more fully hereinafter. For example,lipids bearing targeting glucose moieties may be prepared by reacting,for example, α-glucopyranosyl bromide tetrabenzyl withω-trifluoroacetyl-aminopolyethyleneglycol to obtain ω-glucopyranosyltetrabenzyl-ω'-trifluoroacetyl-aminopolyethyleneglycol. This may then behydrolyzed in a sodium carbonate or potassium carbonate solution andthen hydrogenated to obtain ω-glucopyranpsyl-ω'amino-polyethyleneglycol.Aminoglyco-pyranosyl terminated polyethyleneglycol may then react withN-DPGS-succinimide to form the lipid bearing saccharideDPGS-NH-PEG-Glucose. In certain embodiments, the targeting ligandstarget cancer cells or tumor cells.

In another embodiment, the targeting ligand incorporated into thecompositions of the present invention may be of the formula: ##STR14##where each X₁ is independently --O--, --S--, --SO--, --SO₂ --, --NR₄ --,--X₄ --C(═X₅)--, --C(═X₅)--X₄ -- or --C(═X₅)--; each of X₂ and X₃ isindependently a direct bond, --R₅ --X₄ --C(═X₅)--, --R₅ -C(═X₅)--X₄,--X₄ --C(═X₅)--R₅ --, --C(═X₅)--X₄ --R₅ --, --X₄ --R₅ --C(═X₅)--X₄ --,--R₅ --X₄ --C(═X₅)--R₅ --C(═X₅)--X₄ -- or --R₅ --C(═X₅)--X₄ --R₅ --X₄--C(═X₅)--; each X₄ is independently --O--, --NR₄ -- or --S--; each X₅is independently O or S; M is --R₅ --X₄ --C(═X₅)--, --R₅ --C(═X₅)--X₄--, --R₅ --X₄ --(YX₅)P(═X₅)--X₄ -- or --X₄ --(YX₅)P(═X₅)--X₄ --R₅ --;each n is, independently, 0 or 1; Y is hydrogen or a pharmaceuticallyacceptable counter ion; Z is a hydrophilic polymer; Q is a targetingligand or a precursor to a targeting ligand; each R₁ is independently analkyl group of 1 to about 50 carbons that may optionally be substitutedwith one or more halogen atoms; each R₂ is independently an alkylenegroup of 1 to about 30 carbons that may optionally be substituted withone or more halogen atoms; each of R₃ and R₄ is independently hydrogenor alkyl of 1 to about 10 carbons; and each R₅ is independently a directbond or alkylene of 1 to about 30 carbons.

In the above formula, when any symbol appears more than once in aparticular formula or substituent, its meaning in each instance isindependent of the other. Also in the above formula, it is intended thatwhen each of two or more adjacent symbols is defined as being a "directbond" to provide multiple, adjacent direct bonds, the multiple andadjacent direct bonds devolve into a single direct bond.

In preferred embodiments, each X₁ is independently --X₄ --C(═X₅)--,--C(═X₅)--X₄ -- or --C(═X₅)--. More preferably, each X₁ is independently--X₄ --C(═X₅)-- or --C(═X₅)--X₄ --. Even more preferably, X₁ is--C(═X₅)--X₄ --, for example, --C(═O)--O--.

In preferred embodiments, each of X₂ and X₃ is independently a directbond, --R₅ --X₄ --C(═X₅)--, --R₅ --C(═X₅)--X₄, --X₄ --C(═X₅)--R₅ --,--C(═X₅)--X₄ --R₅ --, --X₄ --R₅ --C(═X₅)--X₄ -- or --R₅ --X₄--C(═X₅)--R₅ --C(═X₅)--X₄ --. More preferably, X₂ is --CH₂ CH₂--C(═O)--NH-- or --CH₂ CH₂ NH--C(═O)--CH,CH₂ --C(═O)--NH-- and X₃ is adirect bond, --C(═O)--NH--, --NH--C(═O)--, --NH--C(═O)--CH₂, --NHCH₂--C(═O)--NH-- or --NH--C(═O)--CH₂ CH₂.

Preferably, each X₄ is independently --O-- or --NR₄ --.

Preferably, X₅ is O.

In certain preferred embodiments, M is --R₅ --X₄ --C(═X₅)-- or --R₅ --X₄--(YX₅)P(═X₅)--X₄ --, with M more preferably being --CH₂ O--C(═O) or--CH₂ O--(HO)P(═O)--O--. In certain other preferred embodiments, M is--R₅ --X₄ --C(═X₅)-- or --R₅ --C(═X₅)--X₄ --. In yet other preferredembodiments, M is --R₅ --X₄ --(YX₅)P(═X₅)--X₄ -- or --X₄--(YX₅)P(═X₅)--X₄ --R₅ --. wherein at least one of X₄ or X₅ is S.

In the above formula, Z is a hydrophilic polymer. Preferably, Z isselected from the group consisting of polyalkyleneoxides, polyvinylalcohol, polyvinylpyrrolidones, polyacrylamides, polymethacrylamides,polyphosphazenes, poly(hydroxyalkylcarboxylic acids) andpolyoxazolidines. More preferably, Z comprises a polyalkyleneoxide. Evenmore preferably, Z is a polyalkyleneoxide selected from the groupconsisting of polyethylene glycol and polypropylene glycol, withpolyethylene glycol being still more preferred. In certain otherpreferred embodiments, Z is a hydrophilic polymer other thanpolyalkylene-oxides, including polyethylene glycol and polypropyleneglycol. The molecular weight of Z may vary, depending, for example, onthe particular end-use of the compounds. Preferably, Z is a polymerhaving a molecular weight which ranges from about 100 to about 10,000,and all combinations and subcombinations of ranges therein. Morepreferably, Z is a polymer having a molecular weight of from about 1,000to about 5,000. Also preferred are polymers which exhibitpolydispersities ranging from greater than about 1 to about 3, and allcombinations and subcombinations of ranges therein. More preferably, Zis a polymer having a polydispersity of from greater than about 1 toabout 2, with polydispersities of from greater than about 1 to about 1.5being even more preferred, and polydispersities of from greater thanabout 1 to about 1.2 being still more preferred.

In the above formula, Q is a targeting ligand or a precursor thereto. Inembodiments where Q is a targeting ligand, Q is preferably selected fromthe group consisting of proteins, peptides, saccharides, steroids,steroid analogs, and genetic material. In these latter embodiments, Q ispreferably selected from the group consisting of proteins, peptides andsaccharides.

In the above formula, each R₁ is independently alkyl which ranges from 1to about 50 carbons, and all combinations and subcombinations of rangestherein, or alkenyl of from about 2 to about 50 carbons, and allcombinations and subcombinations of ranges therein. Preferably, each R₁is independently alkyl of greater than 1 to about 40 carbons. Morepreferably, each R₁ is independently alkyl of about 5 to about 30carbons. Even more preferably, each R₁ is independently alkyl of about10 to about 20 carbons, with alkyl of about 15 carbons being still morepreferred. In certain preferred embodiments, R₁ is a shorter chain alkylof from 1 to about 20 carbons. In certain other preferred embodiments,R₁ is a longer chain alkyl of from about 20 to about 50 carbons, orabout 30 to about 50 carbons. In other preferred embodiments, the alkylgroup in R₁ may be substituted with one or more fluorine atoms, and maybe perfluorinated.

In the above formula, each R₂ is independently alkylene which rangesfrom 1 to about 30 carbons, and all combinations and subcombinations ofranges therein. Preferably, each R₂ is independently alkylene of 1 toabout 20 carbons. More preferably, each R₂ is independently alkylene of1 to about 10 carbons. Even more preferably, each R₂ is independentlyalkylene of 1 to about 5 carbons, with methylene being especiallypreferred. In other preferred embodiments, the alkylene group in R₂ maybe substituted with one or more fluorine atoms, and may beperfluorinated.

In the above formula, each of R₃ and R₄ is independently hydrogen oralkyl which ranges from 1 to about 10 carbons, and all combinations andsubcombinations of ranges therein. Preferably, each of R₃ and R₄ ishydrogen or alkyl of 1 to about 5 carbons. More preferably, each of R₃and R₄ is hydrogen.

In the above formula, each R₅ is independently a direct bond or alkylenewhich ranges from 1 to about 30 carbons, and all combinations andsubcombinations of ranges therein. Preferably, each R₅ is independentlya direct bond or alkylene of 1 to about 20 carbons. More preferably,each R₅ is independently a direct bond or alkylene of 1 to about 10carbons. Even more preferably, each R₅ is independently a direct bond oralkylene of 1 to about 5 carbons. Still more preferably, each R₅ is adirect bond or --(CH₂)_(x) --, where x is 1 or 2.

The compositions of the present invention may be stored in an aqueousmedium and used as preformed compositions prior to use. They may also belyophilized with conventional techniques and cryopreserving agents. Thecompositions can then be rehydrated or reconstituted prior toadministration to a patient. Cryopreserving agents prevent thecompositions from being damaged from ice crystal intercalation duringthe sublimation of water. Agents suitable for cryoprotection includecarbohydrates such as saccharides, such as sucrose, sugar alcohols suchas mannitol and sorbitol, surface active agents such as polyoxyalkylenesorbitan fatty acid esters (such as the class of compounds referred toas TWEEN®, including TWEEN® 20, TWEEN® 40 and TWEEN® 80, commerciallyavailable from ICI Americans, Inc., Wilmington, Del.) and glycerol anddimethylsulfoxide.

A wide variety of methods are available for the preparation of thestabilizing materials and compositions, including vesicles. Includedamong these methods are, for example, shaking, drying, gas-installation,spray drying, and the like. Suitable methods for preparing vesiclecompositions are described, for example, in U.S. Pat. No. 5,469,854, thedisclosure of which is hereby incorporated herein by reference in itsentirety. The compositions are preferably prepared from lipids whichremain in the gel state.

Micelles may be prepared using any of a variety of conventional micellarpreparatory methods which will be apparent to one skilled in the art.These methods typically involve suspension of the stabilizing material,such as a lipid compound, in an organic solvent, evaporation of thesolvent, resuspension in an aqueous medium, sonication andcentrifugation. The foregoing methods, as well as others, are described,for example, in Canfield et al, Methods in Enzymology, 189:418-422(1990); El-Gorab et al, Biochem. Biophys. Acta, 306:58-66 (1973);Colloidal Surfactant, Shinoda, K., Nakagana, Tamamushi and Isejura,Academic Press, NY (1963) (especially "The Formation of Micelles",Shinoda, Chapter 1, pp. 1-88); Catalysis in Micellar and MacromolecularSystems, Fendler and Fendler, Academic Press, NY (1975). The disclosuresof each of the foregoing publications are hereby incorporated herein byreference in their entirety.

In liposomes, the lipid compound(s) may be in the form of a monolayer,bilayer, or multi-layer (i.e., multilamellar) and the monolayer orbilayer lipids may be used to form one or more monolayers or bilayers.In the case of more than one monolayer or bilayer, the monolayers orbilayers are generally concentric. Thus, lipids may be used to formunilamellar compositions or liposomes (comprised of one monolayer orbilayer), oligolamellar compositions or liposomes (comprised of two orthree monolayers or bilayers) or multilamellar compositions or liposomes(comprised of more than three monolayers or bilayers).

Additionally, the compositions may be prepared using any one of avariety of conventional liposomal preparatory techniques which will beapparent to one skilled in the art, including, for example, solventdialysis, French press, extrusion (with or without freeze-thaw), reversephase evaporation, simple freeze-thaw, sonication, chelate dialysis,homogenization, solvent infusion, microemulsification, spontaneousformation, solvent vaporization, solvent dialysis, French pressure celltechnique, controlled detergent dialysis, and others, each involving thepreparation of the compositions in various fashions. See, e.g., Maddenet al., Chemistry and Physics of Lipids, 53:37-46 (1990), the disclosureof which is hereby incorporated herein by reference in its entirety.Suitable freeze-thaw techniques are described, for example, inInternational Application Serial No. PCT/US89/05040, filed Nov. 8, 1989,the disclosure of which is hereby incorporated herein by reference inits entirety. Methods. which involve freeze-thaw techniques arepreferred in connection with the preparation of liposomes. Preparationof the liposomes may be carried out in a solution, such as an aqueoussaline solution, aqueous phosphate buffer solution, or sterile water.The liposomes may also be prepared by various processes which involveshaking or vortexing, which may be achieved, for example, by the use ofa mechanical shaking device, such as a Wig-L-Bug™ (Crescent Dental,Lyons, Ill.), a Mixomat (Degussa AG Frankfurt, Germany), a Capmix (EspeFabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany),a Silamat Plus (Vivadent, Lechtenstein), or a Vibros (Quayle Dental,Sussex, England). Conventional microemulsification equipment, such as aMicrofluidizer™ (Microfluidics, Woburn, Mass.) may also be used.

Spray drying may be employed to prepare gas filled vesicles. Utilizingthis procedure, the stabilizing materials, such as lipids, may bepre-mixed in an aqueous environment and then spray dried to produce gasand/or gaseous precursor filled vesicles. The vesicles may be storedunder a headspace of a desired gas.

Many liposomal preparatory techniques which may be adapted for use inthe preparation of vesicle compositions are discussed, for example, inU.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282,4,310,505, and 4,921,706; U.K. Patent Application GB 2193095A;International Application Serial Nos. PCT/US85/01161 and PCT/US89/05040;Mayer et al., Biochimica et Biophysica Acta, 858:161-168 (1986); Hope etal., Biochimica et Biophysica Acta, 812:55-65 (1985); Mayhew et al.,Methods in Enzymology, 149:64-77 (1987); Mayhew et al., Biochimica etBiophysica Acta, 755:169-74 (1984); Cheng et al, InvestigativeRadiology, 22:47-55 (1987); and Liposome Technology, Gregoriadis, G.,ed., Vol. I, pp. 29-31, 51-67 and 79-108 (CRC Press Inc., Boca Raton,Fla. 1984), the disclosures of each of which are hereby incorporated byreference herein in their entirety.

In connection with stabilizing materials, and especially compositions inthe form of vesicles, it may be advantageous to prepare the compositionsat a temperature below the gel to liquid crystalline phase transitiontemperature of the lipids. This phase transition temperature is thetemperature at which a lipid bilayer will convert from a gel state to aliquid crystalline state. See, for example, Chapman et al., J. Biol.Chem., 249:2512-2521 (1974), the disclosure of which is herebyincorporated by reference herein in its entirety. Generally, vesiclesthat are prepared from lipids that possess higher gel state to liquidcrystalline state phase transition temperatures tend to have enhancedimpermeability at any given temperature. See Derek Marsh, CRC Handbook.of Lipid Bilayers (CRC Press, Boca Raton, Fla. 1990), at p. 139 for mainchain melting transitions of saturateddiacyl-sn-glycero-3-phosphocholines. The gel state to liquid crystallinestate phase transition temperatures of various lipids will be apparentto one skilled in the art and are described, for example, byGregoriadis, ed., Liposome Technology, Vol. I, 1-18 (CRC Press, 1984).

Compositions comprising a gas can be prepared by agitating an aqueoussolution containing, if desired, a stabilizing material, in the presenceof a gas. The term "agitating" means any shaking motion of an aqueoussolution such that gas is introduced from the local ambient environmentinto the aqueous solution. This agitation is preferably conducted at atemperature below the gel to liquid crystalline phase transitiontemperature of the lipid. The shaking involved in the agitation of thesolutions is preferably of sufficient force to result in the formationof a lipid composition, including vesicle compositions, and particularlyvesicle compositions comprising gas filled vesicles. The shaking may beby swirling, such as by vortexing, side-to-side, or up and down motion.Different types of motion may be combined. Also, the shaking may occurby shaking the container holding the aqueous lipid solution, or byshaking the aqueous solution within the container without shaking thecontainer itself.

The shaking may occur manually or by machine. Mechanical shakers thatmay be used include, for example, a shaker table such as a VWRScientific (Cerritos, Calif.) shaker table, as well as any of theshaking devices described hereinbefore, with the Capmix (Espe FabrikPharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany) beingpreferred. It has been found that certain modes of shaking or vortexingcan be used to make vesicles within a preferred size range. Shaking ispreferred, preferably with the Espe Capmix mechanical shaker. With thismethod, it a reciprocating motion is preferably utilized to generate thelipid compositions, particularly vesicles. The reciprocating motion ispreferably in the form of an arc. The rate of reciprocation, as well asthe arc thereof, is particularly important in connection with theformation of vesicles. Preferably, the number of reciprocations or fullcycle oscillations is from about 1000 to about 20,000 per minute, morepreferably from about 2500 to about 8000 per minute, even morepreferably from about 3300 to about 5000 per minute. The number ofoscillations can be dependent upon the mass of the contents beingagitated. Generally, a larger mass requires fewer oscillations. Theaction of gas emitted under high velocity or pressure is another meansfor producing shaking.

It will be understood that with a larger volume of aqueous solution, thetotal amount of force will be correspondingly increased. Vigorousshaking is defined as at least about 60 shaking motions per minute.Preferably, vortexing occurs at about 60 to about 300 revolutions perminute, more preferably at about 300 to about 1800 revolutions perminute.

In addition to the simple shaking methods described above, moreelaborate methods can be employed, including, for example, liquidcrystalline shaking gas instillation processes and vacuum drying gasinstillation processes, such as those described in U.S. Pat. Nos.5,469,854, 5,580,575, 5,585,112, and 5,542,935, and U.S. applicationSer. No. 08/307,305, filed Sep. 16, 1994, the disclosures of which areincorporated herein by reference in their entirety. Emulsion processesmay be employed in the preparation of the compositions of the presentinvention. Such emulsification processes are described, for example, inQuay, U.S. Pat. Nos. 5,558,094, 5,558,853, 5,558,854, and 5,573,751, thedisclosures of which are hereby incorporated herein by reference intheir entirety.

Although any number of techniques can be used, the compositions of thepresent invention are preferably prepared using a shaking technique.Preferably, the shaking technique involves agitation with a mechanicalshaking apparatus, such as an Espe Capmix (Seefeld, Oberay Germany),using, for example, the techniques described in U.S. application Ser.No. 160,232, filed Nov. 30, 1993, the disclosure of which is herebyincorporated herein by reference in its entirety. In addition, afterextrusion and sterilization procedures, which are discussed in detailbelow, agitation or shaking may provide compositions which containsubstantially no or minimal residual anhydrous lipid phase in theremainder of the solution. (Bangham, et al, J. Mol. Biol., 13:238-252(1965).) Other preparatory techniques include those described in Unger,U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporatedherein by reference in its entirety.

Foams can also be produced by shaking gas filled vesicles, wherein thefoam appears on the top of the aqueous solution, and is coupled with adecrease in the volume of the aqueous solution upon the formation offoam. Preferably, the final volume of the foam is at least about twotimes the initial volume of the aqueous stabilizing material solution;more preferably, about three times the initial volume of the aqueoussolution; even more preferably, about four times the initial volume ofthe aqueous solution; and most preferably, all of the aqueousstabilizing material solution is converted to foam.

The required duration of shaking time may be determined by detection ofthe formation of foam. For example, 10 ml of lipid solution in a 50 mlcentrifuge tube may be vortexed for about 15-20 minutes or until theviscosity of the gas filled liposomes becomes sufficiently thick so thatit no longer clings to the side walls as it is swirled. At this time,the foam may cause the solution containing the gas filled liposomes toraise to a level of 30 to 35 ml.

The concentration of lipid required to form a preferred foam level willvary depending upon the type of lipid used, and may be readilydetermined by one skilled in the art, in view of the present disclosure.For example, the concentration of 1,2-dipalmitoyl-phosphatidylcholine(DPPC) used to form gas filled liposomes according to the methods of thepresent invention is about 20 mg/ml to about 30 mg/ml saline solution.The concentration of distearoylphosphatidylcholine (DSPC) used is about5 mg/ml to about 10 mg/ml saline solution. Specifically, DPPC in aconcentration of 20 mg/ml to 30 mg/ml, upon shaking, yields a totalsuspension and entrapped gas volume four times greater than thesuspension volume alone. DSPC in a concentration of 10 mg/ml, uponshaking, yields a total volume completely devoid of any liquidsuspension volume and contains entirely foam.

Microemulsification is a common method of preparing an emulsion of afoam precursor. Temperature increases and/or lowered pressures willcause foaming as gas bubbles form in the liquid. The foam may bestabilized, for example, by surfactants, fluorosurfactants, detergentsor polymers.

The size of gas filled vesicles can be adjusted, if desired, by avariety of procedures, including, for example, microemulsification,vortexing, extrusion, filtration, sonication, homogenization, repeatedfreezing and thawing cycles, extrusion under pressure through pores ofdefined size, and similar methods. Gas filled vesicles prepared by themethods described herein can range in size from less than about 1 μm togreater than about 100 μm. After extrusion and sterilization procedures,which are discussed herein, agitation or shaking provides vesiclecompositions which provide substantially no or minimal residualanhydrous lipid phase in the remainder of the solution. (Bangham et al,J. Mol. Biol., 13:238-252 (1965)). If desired, the compositions of thepresent invention may be used as they are formed, without any attempt atfurther modification of the size thereof For intravascular use, thevesicles preferably have diameters of less than about 30 μm, and morepreferably, less than about 12 μm. For targeted intravascular useincluding, for example, binding to certain tissue, such as canceroustissue, the vesicles can be significantly smaller, for example, lessthan about 100 nm in diameter. For enteric or gastrointestinal use, thevesicles can be significantly larger, for example, up to a millimeter insize. Preferably, the vesicles are sized to have diameters of from about2 μm to about 100 μm.

The gas filled vesicles may be sized by a simple process of extrusionthrough filters wherein the filter pore sizes control the sizedistribution of the resulting gas filled vesicles. By using two or morecascaded or stacked sets of filters, for example, a, 10 μm filterfollowed by an 8 μm filter, the gas filled vesicles can be selected tohave a very narrow size distribution of about 7 to about 9 μm. Afterfiltration, these gas filled vesicles can remain stable for over 24hours.

The sizing or filtration step may be accomplished by the use, forexample, of a filter assembly when the composition is removed from asterile vial prior to use, or more preferably, the filter assembly maybe incorporated into a syringe during use. The method of sizing thevesicles will then comprise using a syringe comprising a barrel, atleast one filter, and a needle; and will be carried out by an extractionstep which comprises extruding the vesicles from the barrel through thefilter fitted to the syringe between the barrel and the needle, therebysizing the vesicles before they are administered to a patient. Theextraction step may also comprise drawing the vesicles into the syringe,where the filter will function in the same way to size the vesicles uponentrance into the syringe. Another alternative is to fill such a syringewith vesicles which have already been sized by some other means, inwhich case the filter now functions to ensure that only vesicles withinthe desired size range, or of the desired maximum size, are subsequentlyadministered by extrusion from the syringe.

In certain preferred embodiments, the vesicle compositions may be heatsterilized or filter sterilized and extruded through a filter prior toshaking. Generally, vesicle compositions comprising a gas may be heatsterilized, and vesicle compositions comprising gaseous precursors maybe filter sterilized. Once gas filled vesicles are formed, they may befiltered for sizing as described above. Performing these steps prior tothe formation of gas and gaseous precursor filled vesicles providesterile gas filled vesicles ready for administration to a patient. Forexample, a mixing vessel, such as a vial or syringe, may be filled witha filtered lipid composition, and the composition may be sterilizedwithin the mixing vessel, for example, by autoclaving. Gas may beinstilled into the composition to form gas filled vesicles by shakingthe sterile vessel. Preferably, the sterile vessel is equipped with afilter positioned such that the gas filled vesicles pass through thefilter before contacting a patient.

The step of extruding the solution of lipid compound through a filterdecreases the amount of unhydrated material by breaking up any driedmaterials and exposing a greater surface area for hydration. Preferably,the filter has a pore size of about 0.1 to bout 5 μm, more preferably,about 0.1 to about 4 μm, even more preferably, about 0.1 o about 2 μm,and still more preferably, about 1 μm. Unhydrated compound, which isgenerally undesirable, appears as amorphous clumps of non-uniform size.

The sterilization step provides a composition that may be administeredto a patient for therapeutic applications, such as drug delivery ordiagnostic imaging. In preferred embodiments, sterilization may beaccomplished by heat sterilization, such as autoclaving the solution ata temperature of at least about 100° C., preferably at about 100° C. toabout 130° C., even more preferably about 110° C. to about 130° C.,still more preferably about 120° C. to about 130° C., and mostpreferably about 130° C. Heating occurs for at least about 1 minute,more preferably about 1 to about 30 minutes, even more preferably about10 to about 20 minutes, and most preferably about 15 minutes. Ifdesired, the extrusion and heating steps may be reversed or only one ofthe two steps can be used. Other modes of sterilization may be used,including, for example, exposure to gamma radiation.

In addition, gaseous precursors contained in vesicles can be formulatedwhich, upon activation, for example, by exposure to elevatedtemperature, varying pH, or light, undergo a phase transition from, forexample, a liquid, including a liquid entrapped in a vesicle, to a gas,expanding to create the gas filled vesicles described herein. Thistechnique is described in U.S. application Ser. No. 08/159,687, filedNov. 30, 1993, and U.S. Pat. No. 5,542,935, the disclosures of which arehereby incorporated herein by reference in their entirety.

The preferred method of activating the gaseous precursor is by exposureto elevated temperature. Activation or transition temperature, and liketerms, refer to the boiling point of the gaseous precursor and is thetemperature at which the liquid to gaseous phase transition of thegaseous precursor takes place. Useful gaseous precursors have boilingpoints in the range of about -100° C. to about 70° C. The activationtemperature is particular to each gaseous precursor. An activationtemperature of about 37° C., or about human body temperature, ispreferred for gaseous precursors. Thus, a liquid gaseous precursor isactivated to become a gas at about 37° C. or lower. The gaseousprecursor may be in liquid or gaseous phase for use in the methods ofthe present invention.

The methods of preparing the gaseous precursor filled vesicles may becarried out below the boiling point of the gaseous precursor such that aliquid is incorporated in the composition. The methods may also beconducted at the boiling point of the gaseous precursor, such that a gasis incorporated, for example, into a vesicle. For gaseous precursorshaving low temperature boiling points, liquid precursors may beemulsified using a microfluidizer device chilled to a low temperature.The boiling points may also be depressed using solvents in liquid mediato utilize a precursor in liquid form. Further, the methods may beperformed where the temperature is increased throughout the process,such that the process starts with a gaseous precursor as a liquid andends with a gas.

The gaseous precursor may be selected to form the gas in situ in thetargeted tissue or fluid, in vivo upon entering the patient or animal,prior to use, during storage, or during manufacture. The methods ofproducing the temperature-activated gaseous precursor filled vesiclesmay be carried out at a temperature below the boiling point of thegaseous precursor. In this embodiment, the gaseous precursor isentrapped within a vesicle such that the phase transition does not occurduring manufacture. Instead, the gaseous precursor filled vesicles aremanufactured in the liquid phase of the gaseous precursor. Activation ofthe phase transition may take place when the temperature is allowed toexceed the boiling point of the precursor. The size of the vesicles,upon attaining the gaseous state, may be determined when the amount ofliquid in a droplet of liquid gaseous precursor is known.

Alternatively, the gaseous precursors may be utilized to create stablegas filled vesicles which are pre-formed prior to use. In thisembodiment, the gaseous precursor is added to a container housing alipid composition at a temperature below the liquid-gaseous phasetransition temperature of the gaseous precursor. As the temperature isincreased, and an emulsion is formed between the gaseous precursor andliquid solution, the gaseous precursor undergoes transition from theliquid to the gaseous state. As a result of this heating and gasformation, the gas displaces the air in the head space above the liquidmixture so as to form gas filled vesicles which entrap the gas of thegaseous precursor, ambient gas (e.g. air), or coentrap gas state gaseousprecursor and ambient air. This phase transition can be used for optimalmixing and formation of the composition. For example, liquidperfluorobutane can be entrapped in the lipid vesicles and, as thetemperature is raised above the boiling point of perfluorobutane (4°C.), perfluorobutane gas is entrapped in the vesicles.

Accordingly, the gaseous precursors may be selected to form gas filledvesicles in vivo or may be designed to produce the gas filled vesiclesin situ, during the manufacturing process, on storage, or at some timeprior to use. A water bath, sonicator or hydrodynamic activation bypulling back the plunger of a syringe against a closed stopcock may beused to activate targeted gas filled vesicles from temperature-sensitivegaseous precursors prior to IV injection.

By preforming the gaseous precursor in the liquid state into an aqueousemulsion, the maximum size of the vesicle may be estimated by using theideal gas law, once the transition to the gaseous state is effectuated.For the purpose of making gas filled vesicles from gaseous precursors,the gas phase is assumed to form instantaneously and substantially nogas in the newly formed vesicle has been depleted due to diffusion intothe liquid, which is generally aqueous in nature. Hence, from a knownliquid volume in the emulsion, an upper size limit of the gas filledvesicle can be predicted.

A mixture of a lipid and a gaseous precursor, containing liquid dropletsof defined size, may be formulated such that upon reaching a specifictemperature, for example, the boiling point of the gaseous precursor,the droplets will expand into gas filled vesicles of defined size. Thedefined size represents an upper limit to the actual size because theideal gas law cannot account for such factors as gas diffusion intosolution, loss of gas to the atmosphere, and the effects of increasedpressure.

The ideal gas law, which can be used for calculating the increase in thevolume of the gas bubbles upon transitioning from liquid to gaseousstates, is PV=nRT, where P is pressure in atmospheres (atm); V is volumein liters (L); n is moles of gas; T is temperature in degrees Kelvin(K); and R is the ideal gas constant (22.4 L-atm/K-mole). With knowledgeof volume, density, and temperature of the liquid in the mixture ofliquids, the amount, for example, in moles, and volume of liquidprecursor may be calculated which, when converted to a gas, will expandinto a vesicle of known volume. The calculated volume will reflect anupper limit to the size of the gas filled vesicle, assuminginstantaneous expansion into a gas filled vesicle and negligiblediffusion of the gas over the time of the expansion.

For stabilization of the precursor in the liquid state in a mixturewhere the precursor droplet is spherical, the volume of the precursordroplet may be determined by the equation: Volume (sphericalvesicle)=4/3 πr³, where r is the radius of the sphere.

Once the volume is predicted, and knowing the density of the liquid atthe desired temperature, the amount of liquid gaseous precursor in thedroplet may be determined. In more descriptive terms, the following canbe applied: V_(gas) =4/3 π(r_(gas))³, by the ideal gas law, PV=nRT,substituting reveals, V_(gas) =nRT/P_(gas) or, (A) n=4/3 [πr_(gas) ³ ]P/RT, where amount n=4/3 [πr_(gas) ³ P/RT]•MW_(n). Converting back to aliquid volume (B) V_(liq) =[4/3 [πr_(gas) ³ ] P/RT]•MW_(n) /D], where Dis the density of the precursor. Solving for the diameter of the liquiddroplet, (C) diameter/2=[3/4π[4/3•[πr_(gas) ³ ] P/RT] MW_(n) /D]^(1/3),which reduces to Diameter=2[[r_(gas) ³ ] P/RT [MW_(n) /D]]^(1/3).

As a further means of preparing compositions of the desired size for usein the methods of the present invention, and with a knowledge of thevolume and especially the radius of the liquid droplets, one can useappropriately sized filters to size the gaseous precursor droplets tothe appropriate diameter sphere.

A representative gaseous precursor may be used to form a vesicle ofdefined size, for example, 10 μm diameter. In this example, the vesicleis formed in the bloodstream of a human being, thus the typicaltemperature would be 37° C. or 310 K. At a pressure of 1 atmosphere andusing the equation in (A), 7.54×10⁻¹⁷ moles of gaseous precursor wouldbe required to fill the volume of a 10 μm diameter vesicle.

Using the above calculated amount of gaseous precursor and1-fluorobutane, which possesses a molecular weight of 76.11, a boilingpoint of 32.5° C. and a density of 0.7789 g/mL at 20° C., furthercalculations predict that 5.74×10⁻¹⁵ grams of this precursor would berequired for a 10 μm vesicle. Extrapolating further, and with theknowledge of the density, equation (B) further predicts that 8.47×10⁻¹⁶mL of liquid precursor is necessary to form a vesicle with an upperlimit of 10 μm.

Finally, using equation (C), a mixture, for example, an emulsioncontaining droplets with a radius of 0.0272 μm or a correspondingdiameter of 0.0544 μm, is formed to make a gaseous precursor filledvesicle with an upper limit of a 10 μm vesicle.

An emulsion of this particular size could be easily achieved by the useof an appropriately sized filter. In addition, as seen by the size ofthe filter necessary to form gaseous precursor droplets of defined size,the size of the filter would also suffice to remove any possiblebacterial contaminants and, hence, can also be used as a sterilefiltration.

This embodiment for preparing gas filled vesicles may be applied to allgaseous precursors activated by temperature. In fact, depression of thefreezing point of the solvent system allows the use of gaseousprecursors which would undergo liquid-to-gas phase transitions attemperatures below 0° C. The solvent system can be selected to provide amedium for suspension of the gaseous precursor. For example, 20%propylene glycol miscible in buffered saline exhibits a freezing pointdepression well below the freezing point of water alone. By increasingthe amount of propylene glycol or adding materials such as sodiumchloride, the freezing point can be depressed even further.

The selection of appropriate solvent systems may be determined byphysical methods as well. When substances, solid or liquid, hereinreferred to as solutes, are dissolved in a solvent, such as water basedbuffers, the freezing point is lowered by an amount that is dependentupon the composition of the solution. Thus, as defined by Wall, one canexpress the freezing point depression of the solvent by the followingequation: Inx_(a) =In (1-x_(b))=ΔH_(fus) R1/T_(o) -1/T), where x_(a) isthe mole fraction of the solvent; x_(b) is the mole fraction of thesolute; ΔH_(fus) is the heat of fusion of the solvent; and T_(o) is thenormal freezing point of the solvent.

The normal freezing point of the solvent can be obtained by solving theequation. If x_(b) is small relative to x_(a), then the above equationmay be rewritten as x^(b) =ΔH_(fus) /R[T-T_(o) /T_(o) T]≈ΔH_(fus)ΔT/RT_(o) ². This equation assumes the change in temperature ΔT is smallcompared to T₂. This equation can be simplified further by expressingthe concentration of the solute in terms of molality, m (moles of soluteper thousand grams of solvent). Thus, the equation can be rewritten asfollows: X_(b) =m/[m+1000/m_(a) ]≈mMa/1000, where Ma is the molecularweight of the solvent. Thus, substituting for the fraction x_(b)ΔT=[M_(a) RT_(o) ² /1000ΔH_(fus) ]m or ΔT=K_(f) m, where K_(f) =M_(a)RT_(o) ² /1000ΔH_(fus). K_(f) is the molal freezing point and is equalto 1.86 degrees per unit of molal concentration for water at oneatmosphere pressure. The above equation may be used to accuratelydetermine the molal freezing point of solutions of gaseous-precursorfilled vesicles. Accordingly, the above equation can be applied toestimate freezing point depressions and to determine the appropriateconcentrations of liquid or solid solute necessary to depress thesolvent freezing temperature to an appropriate value.

Methods of preparing the temperature activated gaseous precursor filledvesicles include:

(a) vortexing and/or shaking an aqueous mixture of gaseous precursor andadditional materials, including, for example, stabilizing materials,thickening agents and/or dispersing agents. Optional variations of thismethod include autoclaving before vortexing or shaking; heating anaqueous mixture of gaseous precursor; venting the vessel containing themixture/suspension; shaking or permitting the gaseous precursor filledvesicle to form spontaneously and cooling down the suspension of gaseousprecursor filled vesicles; and extruding an aqueous suspension ofgaseous precursor through a filter of about 0.22 μm. Alternatively,filtering may be performed during in vivo administration of the vesiclessuch that a filter of about 0.22 μm is employed;

(b) microemulsification where an aqueous mixture of gaseous precursor isemulsified by agitation and heated to form, for example, vesicles priorto administration to a patient;

(c) heating a gaseous precursor in a mixture, with or without agitation,whereby the less dense gaseous precursor filled vesicles float to thetop of the solution by expanding and displacing other vesicles in thevessel and venting the vessel to release air; and

(d) utilizing in any of the above methods a sealed vessel to hold theaqueous suspension of gaseous precursor and maintaining the suspensionat a temperature below the phase transition temperature of the gaseousprecursor, followed by autoclaving to raise the temperature above thephase transition temperature, optionally with shaking, or permitting thegaseous precursor vesicle to form spontaneously, whereby the expandedgaseous precursor in the sealed vessel increases the pressure in thevessel, and cooling down the gas filled vesicle suspension, after whichshaking may also take place.

Freeze drying is useful to remove water and organic materials prior tothe shaking installation method. Drying installation methods may be usedto remove water from vesicles. By pre-entrapping the gaseous precursorin the dried vesicles (i.e. prior to drying) after warming, the gaseousprecursor may expand to fill the vesicle. Gaseous precursors can also beused to fill dried vesicles after they have been subjected to vacuum. Asthe dried vesicles are kept at a temperature below their gel state toliquid crystalline temperature, the drying chamber can be slowly filledwith the gaseous precursor in its gaseous state. For example,perfluorobutane can be used to fill dried vesicles at temperatures above4° C. (the boiling point of perfluorobutane).

Preferred methods for preparing the temperature activated gaseousprecursor filled vesicles comprise shaking an aqueous solution having alipid compound in the presence of a gaseous precursor at a temperaturebelow the liquid state to gas state phase transition temperature of thegaseous precursor. This is preferably conducted at a temperature belowthe gel state to liquid crystalline state phase transition temperatureof the lipid. The mixture is then heated to a temperature above theliquid state to gas state phase transition temperature of the gaseousprecursor which causes the precursor to volatilize and expand. Heatingis then discontinued, and the temperature of the mixture is then allowedto drop below the liquid state to gas state phase transition temperatureof the gaseous precursor. Shaking of the mixture may take place duringthe heating step, or subsequently after the mixture is allowed to cool.Other methods for preparing gaseous precursor filled vesicles caninvolve shaking an aqueous solution of, for example, a lipid and agaseous precursor, and separating the resulting gaseous precursor filledvesicles.

Conventional, aqueous-filled liposomes of the prior art are routinelyformed at a temperature above the phase transition temperature of thelipids used to make them, since they are more flexible and thus usefulin biological systems in the liquid crystalline state. See, Szoka andPapahadjopoulos, Proc. Natl. Acad. Sci. (1978) 75, 4194-4198. Incontrast, the vesicles made according to embodiments described hereinare gaseous precursor filled, which imparts greater flexibility, sincegaseous precursors after gas formation are more compressible andcompliant than an aqueous solution.

The methods contemplated by the present invention provide for shaking anaqueous solution comprising a lipid, in the presence of a temperatureactivatable gaseous precursor. Preferably, the shaking is of sufficientforce such that a foam is formed within a short period of time, such asabout 30 minutes, and preferably within about 20 minutes, and morepreferably, within about 10 minutes. The shaking may involvemicro-emulsifying, microfluidizing, swirling (such as by vortexing),side-to-side, or up and down motion. In the case of the addition ofgaseous precursor in the liquid state, sonication may be used inaddition to the shaking methods set forth above. Further, differenttypes of motion may be combined. Also, the shaking may occur by shakingthe container holding the aqueous lipid solution, or by shaking theaqueous solution within the container without shaking the containeritself Further, the shaking may occur manually or by machine. Mechanicalshakers that may be used include, for example, the mechanical shakersdescribed hereinbefore, with an Espe Capmix (Seefeld, Oberay Germany)being preferred. Another means for producing shaking includes the actionof gaseous precursor emitted under high velocity or pressure.

According to the methods described herein, a gas, such as air, may alsobe provided by the local ambient atmosphere. The local ambientatmosphere can include the atmosphere within a sealed container, as wellas the external environment. Alternatively, for example, a gas may beinjected into or otherwise added to the container having the aqueouslipid solution or into the aqueous lipid solution itself to provide agas other than air. Gases that are lighter than air are generally addedto a sealed container, while gases heavier than air can be added to asealed or an unsealed container. Accordingly, the present inventionincludes co-entrapment of air and/or other gases along with gaseousprecursors.

The gaseous precursor filled vesicles can be used in substantially thesame manner as the gas filled vesicles described herein, once activatedby application to the tissues of a host, where such factors astemperature or pH may be used to cause generation of the gas. It ispreferred that the gaseous precursors undergo phase transitions fromliquid to gaseous states at or near the normal body temperature of thehost, and are thereby activated, for example, by the in vivo temperatureof the host so as to undergo transition to the gaseous phase therein.Alternatively, activation prior to IV injection may be used, forexample, by thermal, mechanical or optical means. This activation canoccur where, for example, the host tissue is human tissue having anormal temperature of about 37° C. and the gaseous precursors undergophase transitions from liquid to gaseous states near 37° C.

In any of the techniques described above for the preparation of lipidcompositions, bioactive agents, targeting ligands and/or counter ionsmay be incorporated with the lipids before, during or after formation ofthe compositions, as would be apparent to one skilled in the art in viewof the present disclosure. For example, the stabilizing materials and/orcompositions may be prepared from a mixture of lipid compounds, counterions, bioactive agents, targeting ligands, and gases and/or gaseousprecursors. In this case, lipid compositions are prepared as describedabove in which the compositions also comprise counter ions, targetingligands, and/or bioactive agents. Thus, for example, micelles can beprepared in the presence of a counter ion, targeting ligand, and/orbioactive agent. In connection with lipid compositions which comprise agas, the preparation can involve, for example, bubbling a gas directlyinto a mixture of the lipid compounds and one or more additionalmaterials, such as counter ions. Alternatively, the lipid compositionsmay be preformed from lipid compounds, counter ions, bioactive agents,targeting ligands, gases and/or gaseous precursors. In the latter case,the counter ion, targeting ligand, and/or bioactive agent is added tothe lipid composition prior to use. For example, an aqueous mixture ofliposomes and gas may be prepared to which the counter ion, targetingligand, and/or bioactive agent is added and which is agitated to providethe liposome composition. The liposome composition can be readilyisolated since the gas, targeting ligand, and/or bioactive agent filledliposome vesicles generally float to the top of the aqueous solution.Excess bioactive agent and/or targeting ligand can be recovered from theremaining aqueous solution.

As one skilled in the art will recognize, any of the stabilizingmaterials and/or compositions may be lyophilized for storage, andreconstituted or rehydrated, for example, with an aqueous medium (suchas sterile water, phosphate buffered solution, or aqueous salinesolution), with the aid of vigorous agitation. Lyophilized preparationsgenerally have the advantage of greater shelf life. To preventagglutination or fusion of the lipids as a result of lyophilization,additives which prevent such fusion or agglutination may be added.Suitable additives include, for example, sorbitol, mannitol, sodiumchloride, glucose, trehalose, polyvinyl-pyrrolidone and poly(ethyleneglycol) (PEG), for example, PEG 400. These and other additives aredescribed in the literature, such as in the U.S. Pharmacopeia, USP XXII,NF XVII, The United States Pharmacopeia, The National Formulary, UnitedStates Pharmacopeial Convention Inc., 12601 Twinbrook Parkway,Rockville, Md. 20852, the disclosure of which is hereby incorporatedherein by reference in its entirety.

The concentration of lipid required to form a desired stabilizedcomposition will vary depending upon the type of lipid used, and may bereadily determined by routine experimentation. The amount of compositionwhich is administered to a patient can vary. Typically, the intravenousdose may be less than about 10 ml for a 70 kg patient, with lower dosesbeing preferred.

Another embodiment of preparing a composition comprises combining atleast one lipid and a gaseous precursor; agitating until gas filledvesicles are formed; adding a bioactive agent and/or targeting ligand tothe gas filled vesicles such that the bioactive agent and/or targetingligand binds to the gas filled vesicle by a covalent bond ornon-covalent bond; and agitating until a composition comprising gasfilled vesicles and a bioactive agent and/or targeting ligand result.Rather than agitating until gas filled vesicles are formed before addingthe bioactive agent and/or targeting ligand, the gaseous precursor mayremain a gaseous precursor until the time of use. That is, the gaseousprecursor is used to prepare the composition and the precursor isactivated in vivo by temperature, for example.

Alternatively, a method of preparing compositions may comprise combiningat least one lipid and a bioactive agent and/or targeting ligand suchthat the bioactive agent and/or targeting ligand binds to the lipid by acovalent bond or non-covalent bond, adding a gaseous precursor andagitating until a composition comprising gas-filled vesicles and abioactive agent and/or targeting ligand result. In addition, the gaseousprecursor may be added and remain a gaseous precursor until the time ofuse. That is, the gaseous precursor is used to prepare the compositionhaving gaseous precursor filled vesicles and a bioactive agent and/ortargeting ligand which result for use in vivo.

Alternatively, the gaseous precursors may be utilized to create stablegas filled vesicles with bioactive agents and/or targeting ligands whichare pre-formed prior to use. In this embodiment, the gaseous precursorand bioactive agent and/or targeting ligand are added to a containerhousing a suspending and/or stabilizing medium at a temperature belowthe liquid-gaseous phase transition temperature of the respectivegaseous precursor. As the temperature is then exceeded, and an emulsionis formed between the gaseous precursor and liquid solution, the gaseousprecursor undergoes transition from the liquid to the gaseous state. Asa result of this heating and gas formation, the gas displaces the air inthe head space above the liquid suspension so as to form gas filledlipid spheres which entrap the gas of the gaseous precursor, ambient gasfor example, air, or coentrap gas state gaseous precursor and ambientair. This phase transition can be used for optimal mixing andstabilization of the composition. For example, the gaseous precursor,perfluorobutane, can be entrapped in the lipid, and as the temperatureis raised beyond 4° C. (boiling point of perfluorobutane), a lipidentrapped fluorobutane gas results. As an additional example, thegaseous precursor fluorobutane, can be suspended in an aqueoussuspension containing emulsifying and stabilizing agents such asglycerol or propylene glycol and vortexed on a commercial vortexer.Vortexing is commenced at a temperature low enough that the gaseousprecursor is liquid and is continued as the temperature of the sample israised past the phase transition temperature from the liquid to gaseousstate. In so doing, the precursor converts to the gaseous state duringthe microemulsification process. In the presence of the appropriatestabilizing agents, stable gas filled vesicles result.

The stabilized vesicle precursors described above can be used in thesame manner as the other stabilized vesicles used in the presentinvention, once activated by application to the tissues of a host, wheresuch factors as temperature or pH may be used to cause generation of thegas. It is preferred that this embodiment is one wherein the gaseousprecursors undergo phase transitions from liquid to gaseous states at ornear the normal body temperature of the host, and are thereby activatedby the temperature of the host tissues so as to undergo transition tothe gaseous phase therein. More preferably still, this method is onewherein the host tissue is human tissue having a normal temperature ofabout 37° C., and wherein the gaseous precursors undergo phasetransitions from liquid to gaseous states near 37° C.

All of the above embodiments involving preparations of the stabilizedgas filled vesicles used in the present invention, may be sterilized byautoclave or sterile filtration if these processes are performed beforeeither the gas instillation step or prior to temperature mediated gasconversion of the temperature sensitive gaseous precursors within thesuspension. Alternatively, one or more anti-bactericidal agents and/orpreservatives may be included in the formulation of the compositions,such as sodium benzoate, quaternary ammonium salts, sodium azide, methylparaben, propyl paraben, sorbic acid, ascorbylpalmitate, butylatedhydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroaceticacid, ethylenediamine, monothioglycerol, potassium benzoate, potassiummetabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, andorganic mercurial salts. Such sterilization, which may also be achievedby other conventional means, such as by irradiation, will be necessarywhere the stabilized microspheres are used for imaging under invasivecircumstances, for example, intravascularly or intraperitoneally. Theappropriate means of sterilization will be apparent to the artisaninstructed by the present description of the stabilized gas filledvesicles and their use. The compositions are generally stored as anaqueous suspension, but in the case of dried or lyophilized vesicles ordried or lyophilized lipid spheres, the compositions may be stored as adried or lyophilized powder ready to be reconstituted or rehydratedprior to use.

In accordance with the present invention, there are provided methods ofimaging a patient, diagnosing the presence of diseased tissue in apatient, delivering a bioactive agent (with or without a targetingligand) to a patient and/or treating a condition in a patient. Theimaging process of the present invention may be carried out byadministering a composition of the invention to a patient, and thenscanning the patient using, for example, ultrasound, computedtomography, and/or magnetic resonance imaging, to obtain visible imagesof an internal region of a patient and/or of any diseased tissue in thatregion. Diagnostic imaging includes promoting the rupture ofcompositions (such as vesicles) via the methods of the presentinvention. For example, ultrasound may be used to visualize thecompositions and verify the location of the compositionss in certaintissue. In addition, ultrasound may be used to promote rupture of thecompositionss once they reach the intended target, including tissueand/or receptor destinations, thus releasing a bioactive agent.

The compositions of the invention may be administered to the patient bya variety of different means, which will vary depending upon theintended application. As one skilled in the art would recognize,administration of the compositions of the present invention can becarried out in various fashions including, for example, topically,including ophthalmic, dermal, ocular and rectal, intrarectally,transdermally, orally, intraperitoneally, parenterally, intravenously,intralymphatically, intratumorly, intramuscularly, interstitially,intra-arterially, subcutaneously, intraocularly, intrasynovially,transepithelially, transdermally, pulmonarily via inhalation,ophthalmically, sublingually, buccally, or nasal inhalation viainsufflation or nebulization.

Preferably, the compositions of the present invention are administeredto a patient as an infusion. "Infusion" refers to intravascular orintra-arterial administration at a rate of, for example, less than about1 cc/second, more preferably less than about 0.5 cc/second or less thanabout 30 cc/minute, even more preferably about 0.1 cc/minute to about 30cc/minute. Varying the rate of infusion is also desirable. For example,infusion may initially be started at a rate of about 1.0 to about 4.0cc/second, followed by a more sustained infusion rate of about 0.1cc/second. The fast infusion rate initially achieves the optimal levelof the stabilizing material and/or vesicle in the blood, while the slowinfusion rate is better tolerated hemodynamically.

The compositions of the present invention are preferably highly activein low concentrations. The amount of composition of the presentinvention to be administered to a patient depends, for example, onwhether a bioactive agent and/or targeting ligand is being used, themethod in which the composition is being administered, and the age, sex,weight and physical condition of the patient. Generally, treatment isinitiated with small dosages, which can then be increased by smallincrements, until the desired effect under the circumstances isachieved. The targeting aspects of the invention enable lower dosages ofthe compositions to be used for therapy, since the effectiveconcentration of the compositions at the therapeutic site remainsundiluted in the body. Additionally, one skilled in the art may rely onreference materials, such as the Physician's Desk Reference, publishedby Medical Economics Company at Montvale, N.J. 07645-1742, to determinethe appropriate amount of a particular bioactive agent, and hence thecorresponding composition of the invention that may be administered to apatient. In accordance with the present invention, compositionscomprising a bioactive agent may be delivered (with or without atargeting ligand) to a patient (e.g., in a region of the patient) forthe purposes, for example, of treating a condition (i.e., a diseasestate, malady, disorder, etc.) in the patient.

Ultrasound mediated targeting and drug release and activation using thecompositions of the present invention is advantageous for treating avariety of different diseases and medical conditions, such as autoimmunediseases, organ transplants, arthritis, and myasthenia gravis. Followingthe systemic administration of the compositions to a patient, ultrasoundmay then be applied to the affected tissue.

Compositions formulated with penetration enhancing agents, known to oneskilled in the art, may be administered transdermally in a patch orreservoir with a permeable membrane applied to the skin. The use ofrupturing ultrasound may increase transdermal delivery of bioactiveagents, including the compositions of the present invention. Further, amechanism may be used to monitor and modulate delivery of thecompositions. For example, diagnostic ultrasound may be used to visuallymonitor the bursting of the gas filled vesicles and modulate drugdelivery and/or a hydrophone may be used to detect the sound of thebursting of the gas filled vesicles and modulate drug delivery.

The delivery of bioactive agents from the compositions of the presentinvention using ultrasound is best accomplished for tissues which have agood acoustic window for the transmission of ultrasonic energy. This isthe case for most tissues in the body such as muscle, the heart, thelungs, the liver and most other vital structures. In the brain, in orderto direct the ultrasonic energy past the skull, a surgical window may benecessary.

Further, the compositions of the present invention are especially usefulfor bioactive agents that may be degraded in aqueous media or uponexposure to oxygen and/or atmospheric air. For example, the vesicles maybe filled with an inert gas such as nitrogen or argon, for use withlabile bioactive agents. Additionally, the gas filled vesicles may befilled with an inert gas and used to encapsulate a labile bioactiveagent for use in a region of a patient that would normally cause thebioactive agent to be exposed to atmospheric air, such as cutaneous andophthalmic applications.

The invention is useful in delivering bioactive agents to a patient'slungs. For pulmonary applications, dried or lyophilized powderedcompositions may be administered via an inhaler. Aqueous suspensions ofliposomes or micelles, preferably gas/gaseous precursor filled, may beadministered via nebulization. Gas filled liposomes of the presentinvention are lighter than, for example, conventional liquid filledliposomes which generally deposit in the central proximal airway ratherthan reaching the periphery of the lungs. It is therefore believed thatthe gas filled liposomes of the present invention may improve deliveryof a bioactive agent to the periphery of the lungs, including theterminal airways and the alveoli. For application to the lungs, the gasfilled liposomes may be applied through nebulization or insufflation.

In applications such as the targeting of the lungs, which are lined withlipids, the bioactive agent may be released upon aggregation of the gasfilled liposomes with the lipids lining the targeted tissue.Additionally, the gas filled liposomes may burst after administrationwithout the use of ultrasound. Thus, ultrasound need not be applied torelease the drug in the above type of administration.

For vascular administration, the compositions are generally injectedinto the venous system as, for example, a gas and/or gaseous precursorcontaining liposome.

It is a further embodiment of this invention in which ultrasoundactivation affords site specific delivery of the compositions.Generally, the gas and/or gaseous precursor containing vesicles areechogenic and visible on ultrasound. Ultrasound can be used to image thetarget tissue and to monitor the drug carrying vesicles as they passthrough the treatment region. As increasing levels of ultrasound areapplied to the treatment region, this breaks apart the vesicles and/orreleases the drug within the treatment region.

Drug release and/or vesicle rupture can be monitored ultrasonically byseveral different mechanisms. As bubbles are destroyed this results ineventual dissolution of the ultrasound signal. Prior to signaldissolution, however, the vehicles provide an initial burst of signal.In other words as increasing levels of ultrasound energy are applied tothe treatment zone containing the vehicles, there is a transientincrease in signal. This transient increase in signal may be recorded atthe fundamental frequency, the harmonic, odd harmonic or ultraharmonicfrequency.

Generally, the delivery systems of the present invention areadministered in the form of an aqueous suspension such as in water or asaline solution (e.g., phosphate buffered saline). Preferably, the wateris sterile. Also, preferably the saline solution is an isotonic salinesolution, although the saline solution may be hypotonic (e.g., about 0.3to about 0.5% NaCl). The solution may also be buffered to provide a pHrange of about pH 5 to about pH 7.4. In addition, sugars, such asdextrose, and/or salts may be included in the media. Other solutionsthat may be used to administer gas filled liposomes include oils, suchas, for example, almond oil, corn oil, cottonseed oil, ethyl oleate,isopropyl myristate, isopropyl palmitate, mineral oil, myristyl alcohol,octyldodecanol, olive oil, peanut oil, persic oil, sesame oil, soybeanoil, squalene and fluorinated oils.

The size of the stabilizing materials and/or vesicles of the presentinvention will depend upon the intended use. With smaller liposomes,resonant frequency ultrasound will generally be higher than for thelarger liposomes. Sizing also serves to modulate resultant liposomalbiodistribution and clearance. In addition to filtration, the size ofthe liposomes can be adjusted, if desired, by procedures known to oneskilled in the art, such as shaking, microemulsification, vortexing,filtration, repeated freezing and thawing cycles, extrusion, extrusionunder pressure through pores of a defined size, sonication,homogenization, the use of a laminar stream of a core of liquidintroduced into an immiscible sheath of liquid. Extrusion under pressurethrough pores of defined size is a preferred method of adjusting thesize of the liposomes. See, U.S. Pat. Nos. 4,728,578, 4,728,575,4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706; U.K. PatentApplication GB 2193095 A; International Applications PCT/US85/01161 andPCT/US89/05040; Mayer et al., Biochimica et Biophysica Acta, 858:161-168(1986); Hope et al., Biochimica et Biophysica Acta, 812:55-65 (1985);Mayhew et al., Methods in Enzymology, 149:64-77 (1987); Mayhew et al.,Biochimica et Biophysica Acta, 755:169-74 (1984); Cheng et al,Investigative Radiology, 22:47-55 (1987); Liposomes Technology,Gregoriadis, ed., Vol. I, pp. 29-108 (CRC Press Inc, Boca Raton, Fla.,1984). The disclosures of each of the foregoing patents, publicationsand patent applications are hereby incorporated by reference herein intheir entirety.

Since vesicle size influences biodistribution, different size vesiclesmay be selected for various purposes. For intravascular applications,the size range is a mean outside diameter between about 30 nm and about10 μm, preferably about 5 μm. More specifically, for intravascularapplications the size of the vesicles is about 10 μm or less in meanoutside diameter, and preferably less than about 7 μm, and morepreferably less than about 5 μm in mean outside diameter. Preferably,the vesicles are no smaller than about 30 nm in mean outside diameter.To provide therapeutic delivery to organs such as the liver and to allowdifferentiation of tumor from normal tissue, smaller vesicles, betweenabout 30 nm and about 100 nm in mean outside diameter, are preferred.For embolization of a tissue such as the kidney or the lung, thevesicles are preferably less than about 200 μm in mean outside diameter.For intranasal, intrarectal or topical administration, the vesicles arepreferably less than about 100 μm in mean outside diameter. Largevesicles, e.g., between 1 and 10 μm in size, will generally be confinedto the intravascular space until they are cleared by phagocytic elementslining the vessels, such as the macrophages and Kupffer cells liningcapillary sinusoids. For passage to the cells beyond the sinusoids,smaller vesicles, for example, less than about 1 μm in mean outsidediameter, e.g., less than about 300 nm in size, may be utilized. Inpreferred embodiments, the vesicles are administered individually,rather than embedded in a matrix, for example.

For intravenous injection, the size of the particle should be under 7μm. For drug delivery to selective sites in vivo, smaller sizes under 2μm, more preferably under 0.5 μm, and even more preferably under 200 nm,is desired. Most preferably the compositions are under 200 nm in sizeand can be as small as about 5 nm to about 10 nm in size. In thecompositions, the lipids covalently bonded to polymers stabilize thelipid compositions into these small structures usable for in vivoapplications.

For in vitro use, such as cell culture applications, the gas and/orgaseous precursor filled vesicles may be added to the cells in culturesand then incubated. Subsequently sonic energy can be applied to theculture media containing the cells and liposomes.

In carrying out the imaging methods of the present invention, thestabilizing materials and vesicle compositions can be used alone, or incombination with targeting ligands, diagnostic agents, therapeuticagents or other agents, including excipients such as flavoring orcoloring materials, which are well-known to one skilled in the art.

In the case of diagnostic applications, such as ultrasound and CT,energy, such as ultrasonic energy, is applied to at least a portion ofthe patient to image the target tissue. A visible image of an internalregion of the patient is then obtained, such that the presence orabsence of diseased tissue can be ascertained. With respect toultrasound, ultrasonic imaging techniques, including second harmonicimaging, and gated imaging, are known in the art, and are described, forexample, in Uhlendorf, IEEE Transactions on Ultrasonics, Ferroelectrics,and Frequency Control, 14(1):70-79 (1994) and Sutherland et al, Journalof the American Society of Echocardiography, 7(5):441-458 (1994), thedisclosures of each of which are hereby incorporated herein by referencein their entirety.

Ultrasound can be used for both diagnostic and therapeutic purposes. Indiagnostic ultrasound, ultrasound waves or a train of pulses ofultrasound may be applied with a transducer. The ultrasound is generallypulsed rather than continuous, although it may be continuous, ifdesired. Thus, diagnostic ultrasound generally involves the applicationof a pulse of echoes, after which, during a listening period, theultrasound transducer receives reflected signals. Harmonics,ultraharmonics or subharmonics may be used. The second harmonic mode maybe beneficially employed, in which the 2x frequency is received, where xis the incidental frequency. This may serve to decrease the signal fromthe background material and enhance the signal from the transducer usingthe targeted contrast media of the present invention which may betargeted to the desired site, for example, blood clots. Other harmonicsignals, such as odd harmonics signals, for example, 3x or 5x, would besimilarly received using this method. Subharmonic signals, for example,x/2 and x/3, may also be received and processed so as to form an image.

In addition to the pulsed method, continuous wave ultrasound, forexample, Power Doppler, may be applied. This may be particularly usefulwhere rigid vesicles are employed. In this case, the relatively higherenergy of the Power Doppler may be made to resonate the vesicles andthereby promote their rupture. This can create acoustic emissions whichmay be in the subharmonic or ultraharmonic range or, in some cases, inthe same frequency as the applied ultrasound. There will be a spectrumof acoustic signatures released in this process and the transducer soemployed may receive the acoustic emissions to detect, for example, thepresence of a clot. In addition, the process of vesicle rupture may beemployed to transfer kinetic energy to the surface, for example of aclot to promote clot lysis. Thus, therapeutic thrombolysis may beachieved during a combination of diagnostic and therapeutic ultrasound.Spectral Doppler may also be employed. In general, the levels of energyfrom diagnostic ultrasound are insufficient to promote the rupture ofvesicles and to facilitate release and cellular uptake of the bioactiveagents. As noted above, diagnostic ultrasound may involve theapplication of one or more pulses of sound. Pauses between pulsespermits the reflected sonic signals to be received and analyzed. Thelimited number of pulses used in diagnostic ultrasound limits theeffective energy which is delivered to the tissue that is being studied.

Higher energy ultrasound, for example, ultrasound which is generated bytherapeutic ultrasound equipment, is generally capable of causingrupture of the vesicle composition. In general, devices for therapeuticultrasound employ from about 10 to about 100% duty cycles, depending onthe area of tissue to be treated with the ultrasound. Areas of the bodywhich are generally characterized by larger amounts of muscle mass, forexample, backs and thighs, as well as highly vascularized tissues, suchas heart tissue, may require a larger duty cycle, for example, up toabout 100%.

In therapeutic ultrasound, continuous wave ultrasound is used to deliverhigher energy levels. For the rupture of vesicles, continuous waveultrasound is preferred, although the sound energy may also be pulsed.If pulsed sound energy is used, the sound will generally be pulsed inecho train lengths of from about 8 to about 20 or more pulses at a time.Preferably, the echo train lengths are about 20 pulses at a time. Inaddition, the frequency of the sound used may vary from about 0.025 toabout 100 megahertz (MHz). In general, frequency for therapeuticultrasound preferably ranges between about 0.75 and about 3 MHz, withfrom about 1 and about 2 MHz being more preferred. In addition, energylevels may vary from about 0.5 Watt (W) per square centimeter (cm²) toabout 5.0 W/cm², with energy levels of from about 0.5 to about 2.5 W/cm²being preferred. Energy levels for therapeutic ultrasound involvinghyperthermia are generally from about 5 W/cm² to about 50 W/cm². Forvery small vesicles, for example, vesicles having a diameter of lessthan about 0.5 μm, higher frequencies of sound are generally preferredbecause smaller vesicles are capable of absorbing sonic energy moreeffectively at higher frequencies of sound. When very high frequenciesare used, for example, greater than about 10 MHz, the sonic energy willgenerally penetrate fluids and tissues to a limited depth only. Thus,external application of the sonic energy may be suitable for skin andother superficial tissues. However, it is generally necessary for deepstructures to focus the ultrasonic energy so that it is preferentiallydirected within a focal zone. Alternatively, the ultrasonic energy maybe applied via interstitial probes, intravascular ultrasound cathetersor endoluminal catheters. Such probes or catheters may be used, forexample, in the esophagus for the diagnosis and/or treatment ofesophageal carcinoma. In addition to the therapeutic uses discussedabove, the present compositions can be employed in connection withesophageal carcinoma or in the coronary arteries for the treatment ofatherosclerosis, as well as the therapeutic uses described, for example,in U.S. Pat. No. 5,149,319, the disclosure of which is herebyincorporated by reference herein in its entirety.

A therapeutic ultrasound device may be used which employs twofrequencies of ultrasound. The first frequency may be x, and the secondfrequency may be 2x. In preferred form, the device would be designedsuch that the focal zones of the first and second frequencies convergeto a single focal zone. The focal zone of the device may then bedirected to the targeted compositions, for example, targeted vesiclecompositions, within the targeted tissue. This ultrasound device mayprovide second harmonic therapy with simultaneous application of the xand 2x frequencies of ultrasound energy. In the case of ultrasoundinvolving vesicles, this second harmonic therapy may provide improvedrupturing of vesicles as compared to ultrasound energy involving asingle frequency. Also, the preferred frequency range may reside withinthe fundamental harmonic frequencies of the vesicles. Lower energy mayalso be used with this device. An ultrasound device which may beemployed in connection with the aforementioned second harmonic therapyis described, for example, by Kawabata, et al., UltrasonicsSonochemistry, 3:1-5 (1996), the disclosure of which is herebyincorporated by reference herein in its entirety.

For use in ultrasonic imaging, preferably, the vesicles of the inventionpossess a reflectivity of greater than 2 dB, more preferably betweenabout 4 dB and about 20 dB. Within these ranges, the highestreflectivity for the vesicles of the invention is exhibited by thelarger vesicles, by higher concentrations of vesicles, and/or whenhigher ultrasound frequencies are employed.

For therapeutic drug delivery, the rupturing of the compositions and/orliposomes of the invention is easily carried out by applying ultrasoundof a certain frequency to the region of the patient where therapy isdesired, after the liposomes have been administered to or have otherwisereached that region, e.g., via delivery with targeting ligands. It hasbeen found that when ultrasound is applied at a frequency correspondingto the peak resonant frequency of the gas filled vesicles, the vesicleswill rupture and release their contents. The peak resonant frequency canbe determined either in vivo or in vitro, but preferably in vivo, byexposing the stabilizing materials or vesicles to ultrasound, receivingthe reflected resonant frequency signals and analyzing the spectrum ofsignals received to determine the peak, using conventional means. Thepeak, as so determined, corresponds to the peak resonant frequency orsecond harmonic.

Preferably, the stabilizing materials and/or vesicle compositions of theinvention have a peak resonant frequency of between about 0.5 MHz andabout 10 MHz. The peak resonant frequency of the gas filled vesicleswill vary depending on the outside diameter and, to some extent, theelasticity or flexibility of the liposomes, with the larger and moreelastic or flexible liposomes having a lower resonant frequency than thesmaller and less elastic or flexible vesicles.

The gas filled vesicles will also rupture when exposed to non-peakresonant frequency ultrasound in combination with a higher intensity(wattage) and duration (time). This higher energy, however, results ingreatly increased heating, which may not be desirable. By adjusting thefrequency of the energy to match the peak resonant frequency, theefficiency of rupture and bioactive agent release is improved,appreciable tissue heating does not generally occur (frequently noincrease in temperature above about 2° C.), and less overall energy isrequired. Thus, application of ultrasound at the peak resonantfrequency, while not required, is most preferred.

For diagnostic or therapeutic ultrasound, any of the various types ofdiagnostic ultrasound imaging devices may be employed in the practice ofthe invention, the particular type or model of the device not beingcritical to the method of the invention Also suitable are devicesdesigned for administering ultrasonic hyperthermia, as described in U.S.Pat. Nos. 4,620,546, 4,658,828, and 4,586,512, the disclosures of eachof which are hereby incorporated herein by reference in their entirety.Preferably, the device employs a resonant frequency (RF) spectralanalyzer. The transducer probes may be applied externally or may beimplanted. Ultrasound is generally initiated at lower intensity andduration, and then intensity, time, and/or resonant frequency increaseduntil the vesicle is visualized on ultrasound (for diagnostic ultrasoundapplications) or ruptures (for therapeutic ultrasound applications).

Although application of the various principles will be apparent to oneskilled in the art in view of the present disclosure, by way of generalguidance, for gas filled vesicles of about 1.5 to about 10 μm in meanoutside diameter, the resonant frequency will generally be in the rangeof about 1 to about 10 MHz. By adjusting the focal zone to the center ofthe target tissue (e.g., the tumor) the gas filled vesicles can bevisualized under real time ultrasound as they accumulate within thetarget tissue. Using the 7.5 MHz curved array transducer as an example,adjusting the power delivered to the transducer to maximum and adjustingthe focal zone within the target tissue, the spatial peak temporalaverage (SPTA) power will then be a maximum of approximately 5.31 mW/cm²in water. This power will cause some release of the bioactive agent fromthe gas filled vesicles, but much greater release can be accomplished byusing higher power.

By switching the transducer to the doppler mode, higher power outputsare available, up to 2.5 W/cm² from the same transducer. With themachine operating in doppler mode, the power can be delivered to aselected focal zone within the target tissue and the gas filled vesiclescan be made to release the bioactive agent. Selecting the transducer tomatch the resonant frequency of the gas filled vesicles will make therelease of the bioactive agent even more efficient.

For larger diameter gas filled vesicles, e.g., greater than 3 μm in meanoutside diameter, a lower frequency transducer may be more effective inaccomplishing therapeutic release. For example, a lower frequencytransducer of 3.5 MHz (20 mm curved array model) may be selected tocorrespond to the resonant frequency of the gas filled vesicles. Usingthis transducer, 101.6 mW/cm² may be delivered to the focal spot, andswitching to doppler mode will increase the power output (SPTA) to 1.02Wcm².

To use the phenomenon of cavitation to release and/or activate the gasfilled stabilizing materials and/or vesicles, lower frequency energiesmay be used, as cavitation occurs more effectively at lower frequencies.Using a 0.757 MHz transducer driven with higher voltages (as high as 300volts) cavitation of solutions of gas-filled liposomes will occur atthresholds of about 5.2 atmospheres.

The ranges of energies transmitted to tissues from diagnostic ultrasoundon commonly used instruments such as the Piconics Inc. (Tyngsboro,Mass.) Portascan general purpose scanner with receiver pulser 1966 Model661; the Picker (Cleveland, Ohio.) Echoview 8L Scanner including 80CSystem or the Medisonics (Mountain View, Calif.) Model D-9 VersatoneBidirectional Doppler, is known in the art and described, for example,by Carson et al., Ultrasound in Med. & Biol., 3:341-350 (1978), thedisclosure of which is hereby incorporated herein by reference in itsentirety In general, these ranges of energies employed in pulserepetition are useful for diagnosis and monitoring gas-filled liposomesbut are insufficient to rupture the gas-filled liposomes of the presentinvention.

Either fixed frequency or modulated frequency ultrasound may be used.Fixed frequency is defined wherein the frequency of the sound wave isconstant over time. A modulated frequency is one in which the wavefrequency changes over time, for example, from high to low (PRICH) orfrom low to high (CHIRP). For example, a PRICH pulse with an initialfrequency of 10 MHz of sonic energy is swept to 1 MHz with increasingpower from 1 to 5 watts. Focused, frequency modulated, high energyultrasound may increase the rate of local gaseous expansion within theliposomes and rupturing to provide local delivery of bioactive agents.

The frequency of the sound used may vary from about 0.025 to about 100MHz. Frequency ranges between about 0.75 and about 3 MHz are preferredand frequencies between about 1 and about 2 MHz are most preferred.Commonly used therapeutic frequencies of about 0.75 to about 1.5 MHz maybe used. Commonly used diagnostic frequencies of about 3 to about 7.5MHz may also be used. For very small vesicles, e.g., below 0.5 μm inmean outside diameter, higher frequencies of sound may be preferred asthese smaller vesicles will absorb sonic energy more effectively athigher frequencies of sound. When very high frequencies are used, e.g.,over 10 MHz, the sonic energy will generally have limited depthpenetration into fluids and tissues. External application may bepreferred for the skin and other superficial tissues, but for deepstructures, the application of sonic energy via interstitial probes orintravascular ultrasound catheters may be preferred.

Where the gas filled stabilizing materials and/or vesicles are used fordrug delivery, the bioactive agent to be delivered may be embeddedwithin the wall(s) of the vesicle, encapsulated in the vesicle, attachedto the surface of the vesicle and/or any combination thereof. The phrase"attached to" or variations thereof means that the bioactive agent islinked in some manner to the inside and/or the outside wall of thevesicle, such as through a covalent or ionic bond or other means ofchemical or electrochemical linkage or interaction. The phrase"encapsulated in" or variations thereof as used in connection with thelocation of the bioactive agent denotes that the bioactive agent islocated in the internal vesicle void. The phrase "embedded within" orvariations thereof signifies the positioning of the bioactive agentwithin the vesicle wall(s) or layer(s). The phrase "comprising abioactive agent" denotes all of the varying types of therapeuticpositioning in connection with the vesicle. Thus, the bioactive agentcan be positioned variably, such as, for example, entrapped within theinternal void of the gas filled vesicle, situated between the gas andthe internal wall of the gas filled vesicle, incorporated onto theexternal surface of the gas filled vesicle, enmeshed within the vesiclestructure itself and/or any combination thereof. The compositionss mayalso be designed so that there is a symmetric or an asymmetricdistribution of the drug both inside and outside of the stabilizingmaterial and/or vesicle.

More than one bioactive agent may be administered to a patient using thevesicles. For example, a single vesicle may contain more than onebioactive agent or vesicles containing different bioactive agents may beco-administered. Preferably, a targeting ligand is used in conjunctionwith a bioactive agent. For example, a monoclonal antibody capable ofbinding to melanoma antigen and an oligonucleotide encoding at least aportion of IL-2 may be administered at the same time. The phrase "atleast a portion of" means that the entire gene need not be representedby the oligonucleotide, so long as the portion of the gene representedprovides an effective block to gene expression.

Genetic materials and bioactive agents may be incorporated into theinternal gas filled space of these vesicles during the gas installationprocess or into or onto the vesicle membranes of the compositions.Genetic materials and bioactive agents with a high octanol/waterpartition coefficient may be incorporated directly into the layer orwall surrounding the gas but incorporation onto the surface of the gasfilled vesicles is more preferred. To accomplish this, groups capable ofbinding genetic materials or bioactive agents are generally incorporatedinto the stabilizing material layers which will then bind thesematerials. In the case of genetic materials (DNA, RNA, both singlestranded and double stranded and anti-sense and sense oligonucleotides)this is readily accomplished through the use of the compositions of thepresent invention.

Preferred therapeutics include genetic material such as nucleic acids,RNA, and DNA, of either natural or synthetic origin, includingrecombinant RNA and DNA and antisense RNA and DNA. Types of geneticmaterial that may be used include, for example, genes carried onexpression vectors such as plasmids, phagemids, cosmids, yeastartificial chromosomes (YACs), and defective or "helper" viruses,antigene nucleic acids, both single and double stranded RNA and DNA andanalogs thereof, such as phosphoro-thioate and phosphorodithioateoligodeoxynucleotides. Further, the genetic material may be combined,for example, with proteins or other polymers. Genetic materials that maybe applied using the compositions of the present invention include, forexample, DNA encoding at least a portion of an HLA gene, DNA encoding atleast a portion of dystrophin, DNA encoding at least a portion of CFTR,DNA encoding at least a portion of IL-2, DNA encoding at least a portionof TNF, an antisense oligonucleotide capable of binding DNA encoding atleast a portion of Ras.

DNA encoding certain proteins may be used in the treatment of manydifferent types of diseases. For example, adenosine deaminase may beprovided to treat ADA deficiency; tumor necrosis factor and/orinterleukin-2 may be provided to treat advanced cancers; HDL receptormay be provided to treat liver disease; thymidine kinase may be providedto treat ovarian cancer, brain tumors, or HIV infection; HLA-B7 may beprovided to treat malignant melanoma; interleukin-2 may be provided totreat neuroblastoma, malignant melanoma, or kidney cancer; interleukin-4may be provided to treat cancer; HIV env may be provided to treat HIVinfection; antisense ras/p53 may be provided to treat lung cancer; andFactor VIII may be provided to treat Hemophilia B. See, for example,Science, 258, 744-746.

A gas filled vesicle filled with oxygen gas should create extensive freeradicals with cavitation. Also, metal ions from the transition series,especially manganese, iron and copper can increase the rate of formationof reactive oxygen intermediates from oxygen. By encapsulating metalions within the vesicles, the formation of free radicals in vivo can beincreased. These metal ions may be incorporated into the liposomes asfree salts, as complexes, e.g., with EDTA, DTPA DOTA or desferrioxamine,or as oxides of the metal ions. Additionally, derivatized complexes ofthe metal ions may be bound to lipid head groups, or lipophiliccomplexes of the ions may be incorporated into a lipid bilayer, forexample. When exposed to thermal stimulation, e.g., cavitation, thesemetal ions then will increase the rate of formation of reactive oxygenintermediates. Further, radiosensitizers such as metronidazole andmisonidazole may be incorporated into the gas filled vesicles to createfree radicals on thermal stimulation.

As discussed above, the compositions and stabilizing materials of thepresent invention may be used in connection with diagnostic imaging,therapeutic imaging and drug delivery, including, for example,ultrasound (US), magnetic resonance imaging (MRI), nuclear magneticresonance (NMR), computed tomography (CT), electron spin resonance(ESR), nuclear medical imaging, optical imaging, elastography,radiofrequency (RF) and microwave laser. The compositions andstabilizing materials of the present invention may be used incombination with various contrast agents, including conventionalcontrast agents, which may serve to increase their effectiveness ascontrast agents for diagnostic and therapeutic imaging.

Examples of suitable contrast agents for use with MRI in combinationwith the present stabilizing materials include, for example, stable freeradicals, such as, stable nitroxides, as well as compounds comprisingtransition, lanthanide and actinide elements, which may, if desired, bein the form of a salt or may be covalently or non-covalently bound tocomplexing agents, including lipophilic derivatives thereof, or toproteinaceous macromolecules. Preferable transition, lanthanide andactinide elements include, for example, Gd(III), Mn(II), Cu(II),Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II), Eu(III) and Dy(III).More preferably, the elements may be Gd(III), Mn(II), Cu(II), Fe(II),Fe(III), Eu(III) and Dy(III), most preferably Mn(II) and Gd(III). Theforegoing elements may be in the form of a salt, including inorganicsalts, such as a manganese salt, for example, manganese chloride,manganese carbonate, manganese acetate, and organic salts, such asmanganese gluconate and manganese hydroxyl- apatite. Other exemplarysalts include salts of iron, such as iron sulfides, and ferric salts,such as ferric chloride.

The above elements may also be bound, for example, through covalent ornoncovalent association, to complexing agents, including lipophilicderivatives thereof, or to proteinaceous macromolecules. Preferablecomplexing agents include, for example, diethylenetriaminepentaaceticacid (DTPA), ethylenediaminetetraacetic acid (EDTA),1,4,7,10-tetraazacyclododecane-N,N',N',N'"-tetraacetic acid (DOTA),1,4,7,10-tetraaza-cyclododecane-N,N',N'"-triacetic acid (DOTA),3,6,9-triaza-12-oxa-3,6,9-tricarboxy-methylene-10-carboxy-13-phenyltridecanoicacid (B-19036), hydroxybenzylethylene-diamine diacetic acid (HBED),N,N'-bis(pyridoxyl-5-phosphate)ethylene diamine, N,N'-diacetate (DPDP),1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA),1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA),kryptands (macrocyclic complexes), and desferrioxamine. More preferably,the complexing agents are EDTA, DTPA, DOTA, DO3A and kryptands, mostpreferably DTPA. Preferable lipophilic complexes include alkylatedderivatives of the complexing agents EDTA, DOTA, for example,N,N'-bis-(carboxydecylamidomethyl-N-2,3-dihydroxypropyl)ethylenediamine-N,N'-diacetate(EDTA-DDP);N,N'-bis-(carboxyoctadecylamidomethyl-N-2,3-dihydroxy-propyl)ethylenediamine-N,N'-diacetate(EDTA-ODP); andN,N'-Bis(carboxylaurylamido-methyl-N-2,3-dihydroxypropyl)ethylenediamine-N,N'-diacetate(EDTA-LDP); including those described in U.S. Pat. No. 5,312,617, thedisclosure of which is hereby incorporated. herein by reference in itsentirety. Preferable proteinaceous macromolecules include, for example,albumin, collagen, polyarginine, polylysine, polyhistidine, γ-globulinand β-globulin, with albumin, polyarginine, polylysine, andpolyhistidine being more preferred. Suitable complexes therefore includeMn(II)-DTPA, Mn(II)-EDTA, Mn(II)-DOTA, Mn(II)-DO3A, Mn(II)-kryptands,Gd(III)-DTPA, Gd(III)-DOTA, Gd(III)-D03A, Gd(III)-kryptands,Cr(III)-EDTA, Cu(II)-EDTA, or iron-desferrioxamine, more preferablyMn(II)-DTPA or Gd(III)-DTPA.

Nitroxides are paramagnetic contrast agents which increase both T1 andT2 relaxation rates on MRI by virtue of the presence of an unpairedelectron in the nitroxide molecule. As known to one of ordinary skill inthe art, the paramagnetic effectiveness of a given compound as an MRIcontrast agent may be related, at least in part, to the number ofunpaired electrons in the paramagnetic nucleus or molecule, andspecifically, to the square of the number of unpaired electrons. Forexample, gadolinium has seven unpaired electrons whereas a nitroxidemolecule has one unpaired electron. Thus, gadolinium is generally a muchstronger MRI contrast agent than a nitroxide. However, effectivecorrelation time, another important parameter for assessing theeffectiveness of contrast agents, confers potential increased relaxivityto the nitroxides. When the tumbling rate is slowed, for example, byattaching the paramagnetic contrast agent to a large molecule, it willtumble more slowly and thereby more effectively transfer energy tohasten relaxation of the water protons. In gadolinium, however, theelectron spin relaxation time is rapid and will limit the extent towhich slow rotational correlation times can increase relaxivity. Fornitroxides, however, the electron spin correlation times are morefavorable and tremendous increases in relaxivity may be attained byslowing the rotational correlation time of these molecules. The gasfilled vesicles of the present invention are ideal for attaining thegoals of slowed rotational correlation times and resultant improvementin relaxivity. Although not intending to be bound by any particulartheory of operation, it is contemplated that since the nitroxides may bedesigned to coat the perimeters of the vesicles, for example, by makingalkyl derivatives thereof, the resulting correlation times can beoptimized. Moreover, the resulting contrast medium of the presentinvention may be viewed as a magnetic sphere, a geometric configurationwhich maximizes relaxivity.

Exemplary superparamagnetic contrast agents suitable for use with MRI inthe compositions of the present invention include metal oxides andsulfides which experience a magnetic domain, ferro- or ferrimagneticcompounds, such as pure iron, magnetic iron oxide, such as magnetite,γ-Fe₂ O₃, Fe₃ O₄, manganese ferrite, cobalt ferrite and nickel ferrite.Paramagnetic gases can also be employed in the present compositions,such as oxygen 17 gas (¹⁷ O₂). In addition, hyperpolarized xenon, neon,or helium gas may also be employed. Magnetic resonance (MR) whole bodyimaging may then be employed to rapidly screen the body, for example,for thrombosis, and ultrasound may be applied, if desired, to aid inthrombolysis.

The contrast agents, such as the paramagnetic and superparamagneticcontrast agents described above, may be employed as a component withinthe compositionss and/or stabilizing materials. With respect tovesicles, the contrast agents may be entrapped within the internal voidthereof, administered as a solution with the vesicles;, incorporatedwith any additional stabilizing materials, or coated onto the surface ormembrane of the vesicle. Mixtures of any one or more of the paramagneticagents and/or superparamagnetic agents in the present compositions maybe used. The paramagnetic and superparamagnetic agents may also becoadministered separately, if desired.

If desired, the paramagnetic or superparamagnetic agents may bedelivered as alkylated or other derivatives incorporated into thecompositions, especially the lipidic walls of the vesicles. Inparticular, the nitroxides 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, freeradical and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical, can formadducts with long chain fatty acids at the positions of the ring whichare not occupied by the methyl groups via a variety of linkages,including, for example, an acetyloxy linkage. Such adducts are veryamenable to incorporation into the lipid and/or vesicle compositions ofthe present invention.

The stabilizing materials and/or vesicles of the present invention, andespecially the vesicles, may serve not only as effective carriers of thesuperparamagnetic agents described above, but also may improve theeffect of the susceptibility contrast agents. Superparamagnetic contrastagents include metal oxides, particularly iron oxides but includingmanganese oxides, and as iron oxides, containing varying amounts ofmanganese, cobalt and nickel which experience a magnetic domain. Theseagents are nano or microparticles and have very high bulksusceptibilities and transverse relaxation rates. The larger particles,for example, particles having diameters of about 100 nm, have muchhigher R2 relaxivities as compared to R1 relaxivities. The smallerparticles, for example, particles having diameters of about 10 to about15 nm, have somewhat lower R2 relaxivities, but much more balanced R1and R2 values. Much smaller particles, for example, monocrystalline ironoxide particles having diameters of about 3 to about 5 nm, have lower R2relaxivities, but probably the most balanced R1 and R2 relaxation rates.Ferritin can also be formulated to encapsulate a core of very highrelaxation rate superparamagnetic iron. It has been discovered that thelipid and/or vesicle compositions, especially vesicle compositions,including gas filled vesicles, can increase the efficacy and safety ofthese conventional iron oxide based MRI contrast agents.

The iron oxides may simply be incorporated into the stabilizingmaterials and/or vesicles. Preferably, in the case of vesiclesformulated from lipids, the iron oxides may be incorporated into thewalls of the vesicles, for example, by being adsorbed onto the surfacesof the vesicles, or entrapped within the interior of the vesicles.

Without being bound to any particular theory or theories of operation,it is believed that the vesicles of the present invention increase theefficacy of the superparamagnetic contrast agents by several mechanisms.First, it is believed that the vesicles function to increase theapparent magnetic concentration of the iron oxide particles. Also, it isbelieved that the vesicles increase the apparent rotational correlationtime of the MRI contrast agents, including paramagnetic andsuperparamagnetic agents, so that relaxation rates are increased. Inaddition, the vesicles appear to increase the apparent magnetic domainof the contrast medium according to the manner described hereinafter.

Certain of the vesicles of the present invention, and especiallyvesicles formulated from lipids, may be visualized as flexible sphericaldomains of differing susceptibility from the suspending medium,including, for example, the aqueous suspension of the contrast medium orblood or other body fluids, for example, in the case of intravascularinjection or injection into other body locations. In the case offerrites or iron oxide particles, it should be noted that the contrastprovided by these agents is dependent on particle size. This phenomenonis very common and is often referred to as the "secular" relaxation ofthe water molecules. Described in more physical terms, this relaxationmechanism is dependent upon the effective size of the molecular complexin which a paramagnetic atom, or paramagnetic molecule, or molecules,may reside. One physical explanation may be described in the followingSolomon-Bloembergen equations which define the paramagneticcontributions as a function of the T₁ and T₂ relaxation times of a spin1/2 nucleus with gyromagnetic ratio g perturbed by a paramagnetic ion:1/T₁ M=(2/15) S(S+1)γ² g² β² /r⁶ [3τ_(c) /(1+ω_(I) ² τ_(c) ²)+7τ_(c)/(1+ω_(s) ² τ_(c) ²)]+(2/3) S(S+1) A² /h² [τ_(c) /(1+ω_(s) 2τ_(c) ²)]and 1/T₂ M=(1/15) S(S+1) γ² g² β² /r⁶ [4τ_(c) +3τc/(1+ω_(I) ² τ_(c)²)+13τ_(c) /(1+w_(s) ² τ_(c) ²)]+(1/3) S(S+1)A² /h² [τ_(c) /(1+ω_(s)2τ_(c) ²)], where S is the electron spin quantum number; g is theelectronic g factor; β is the Bohr magneton; ω_(I) and ω_(s) (657 w_(I))is the Larmor angular precession frequencies for the nuclear spins andelectron spins; r is the ion-nucleus distance; A is the hyperfinecoupling constant; τ_(c) and τ_(e) are the correlation times for thedipolar and scalar interactions, respectively; and h is Planck'sconstant.

A few large particles may have a much greater effect than a largernumber of much smaller particles, primarily due to a larger correlationtime. If one were to make the iron oxide particles very large however,increased toxicity may result, and the lungs may be embolized or thecomplement cascade system may be activated. Furthermore, it is believedthat the total size of the particle is not as important as the diameterof the particle at its edge or outer surface. The domain ofmagnetization or susceptibility effect falls off exponentially from thesurface of the particle. Generally speaking, in the case of dipolar(through space) relaxation mechanisms, this exponential fall offexhibits an r⁶ dependence for a paramagnetic dipole-dipole interaction.Interpreted literally, a water molecule that is 4 angstroms away from aparamagnetic surface will be influenced 64 times less than a watermolecule that is 2 angstroms away from the same paramagnetic surface.The ideal situation in terms of maximizing the contrast effect would beto make the iron oxide particles hollow, flexible and as large aspossible. It has not been possible to achieve this heretofore and it isbelieved that the benefits have been unrecognized heretofore also. Bycoating the inner or outer surfaces of the vesicles with the contrastagents, even though the individual contrast agents, for example, ironoxide nanoparticles or paramagnetic ions, are relatively smallstructures, the effectiveness of the contrast agents may be greatlyenhanced. In so doing, the contrast agents may function as aneffectively much larger sphere wherein the effective domain ofmagnetization is determined by the diameter of the vesicle and ismaximal at the surface of the vesicle. These agents afford the advantageof flexibility, namely, compliance. While rigid vesicles might lodge inthe lungs or other organs and cause toxic reactions, these flexiblevesicles slide through the capillaries much more easily.

In contrast to the flexible vesicles described above, it may bedesirable, in certain circumstances, to formulate vesicles fromsubstantially impermeable polymeric materials including, for example,polymethyl methacrylate. This would generally result in the formation ofvesicles which may be substantially impermeable and relatively inelasticand brittle. In embodiments involving diagnostic imaging, for example,ultrasound, contrast media which comprise such brittle vesicles wouldgenerally not provide the desirable reflectivity that the flexiblevesicles may provide. However, by increasing the power output onultrasound, the brittle microspheres can be made to rupture, therebycausing acoustic emissions which can be detected by an ultrasoundtransducer.

Nuclear Medicine Imaging (NMI) may also be used in connection with thediagnostic and therapeutic method aspects of the present invention. Forexample, NMI may be used to detect radioactive gases, such as Xe¹³³,which may be incorporated in the present compositions in addition to, orinstead of, the gases discussed above. Such radioactive gases may beentrapped within vesicles for use in detecting, for example, thrombosis.Preferably, bifunctional chelate derivatives are incorporated in thewalls of vesicles, and the resulting vesicles may be employed in bothNMI and ultrasound. In this case, high energy, high quality nuclearmedicine imaging isotopes, such as technetium^(99m) or indium¹¹¹ can beincorporated in the walls of vesicles. Whole body gamma scanning camerascan then be employed to rapidly localize regions of vesicle uptake invivo. If desired, ultrasound may also be used to confirm the presence,for example, of a clot within the blood vessels, since ultrasoundgenerally provides improved resolution as compared to nuclear medicinetechniques. NMI may also be used to screen the entire body of thepatient to detect areas of vascular thrombosis, and ultrasound can beapplied to these areas locally to promote rupture of the vesicles andtreat the clot.

For optical imaging, optically active gases, such as argon or neon, maybe incorporated in the present compositions. In addition, opticallyactive materials, for example, fluorescent materials, includingporphyrin derivatives, may also be used. Elastography is an imagingtechnique which generally employs much lower frequency sound, forexample, about 60 kHz, as compared to ultrasound which can involvefrequencies of over 1 MHz. In elastography, the sound energy isgenerally applied to the tissue and the elasticity of the tissue maythen be determined. In connection with preferred embodiments of theinvention, which involve highly elastic vesicles, the deposition of suchvesicles onto, for example, a clot, increases the local elasticity ofthe tissue and/or the space surrounding the clot. This increasedelasticity may then be detected with elastography. If desired,elastography can be used in conjunction with other imaging techniques,such as MRI and ultrasound.

EXAMPLES

The invention is further demonstrated in the following examples.Examples 1-12 and 15 are actual examples, and Examples 13, 14 and 16-18are prophetic examples. The examples are for purposes of illustrationonly and are not intended to limit the scope of the present invention.

As shown in Examples 1-5, the amount of lipid covalently bonded to thepolymer can be used to influence the size distribution of the lipidcompositions. The size of the resulting compositions depends upon thecontents including the presence of exogenous Ca²⁺. Graphicalrepresentations of the sizing data is shown in FIG. 2-4.

Example 1

Compositions containing varying amounts of dimyristoyl were prepared.Dry lipids were weighed out and hydrated in dH₂ O by heating andstirring in the mole percentage ratio of from 65 mole % to 75 mole %dimyristoylphosphatidylcholine (DMPC); from 15 mole % to 25 mole %dimyristoylphosphatidic acid (DMPA); and from 0 to 20 mole %dimyristoylphosphatidylethanolamine-polyethylene glycol-5,000(DMPE-PEG5,000). A small vial was rinsed with dH2O and tared on aMettler AJ100 (Mettler Instrument Corp. PO Box 71, Hightstown, N.J.,08520) balance and five to ten milligrams of the lyopholized lipid blendwas weighed into the vial. Triple 0.22 μm filtered dI H₂ O was added byweight until the final concentration of lipid was 1 mg/ml. This mixturewas heated to 45-50° C. for 1 hour and then sonicated in a Aquasoniccleaner Model 75 HT (VWR) at room temperature for 1 hour in 30 minuteincrements to prevent excessive heating. This treatment reduced therelative size of the lipid particles in solution. The particles weresized using a NICOMP C370 (Particle Sizing Systems, 75 Aero Camino SuiteB, Santa Barbara Calif. 93117) by a modified NNLS/CONTIN algorithmresulting in a multimodal size distribution with peak intensitydiameters plotted as shown in FIG. 2A. These compositions may beconsidered controls since no divalent cations were added either duringformation or resusupension of the lipids.

Example 2

Compositions containing varying amounts of dipalmitoyl and Ca²⁺ in theresuspending media were prepared. Dry lipids were weighed out andhydrated in dI H₂ O by heating and stirring in the mole percentage ratioof from 65 mol % to 75 mol % dipalmitoylphosphatidylcholine (DPPC); from15 mol % to 25 mol % dipalmitoylphosphatidic acid (DPPA) and from 0 to20 mol % dipalmitoylphosphatidylethanolamine-polyethylene glycol-5,000(DPPE-PEG5,000). A small vial was rinsed with dI H₂ O and tared on aMettler AJ100 (Mettler Instrument Corp. PO Box 71, Hightstown, N.J.,08520) balance and five to ten milligrams of the lyopholized lipid blendwas weighed into the vial. Triple 0.22 μm filtered 10 mM CaCl₂ was addedby weight until the final concentration of lipid was 1 mg/ml. Thismixture was heated to 45-50° C. for 1 hour and then sonicated in aAquasonic cleaner Model 75 HT (VWR) at room temperature for 1 hour in 30minute increments to prevent excessive heating. This treatment reducedthe relative size of the lipid particles in solution. The particles weresized using a NICOMP C370 (Particle Sizing Systems, 75 Aero Camino SuiteB, Santa Barbara Calif. 93117) by a modified NNLS/CONTIN algorithmresulting in a multimodal size distribution with peak intensitydiameters as plotted in FIG. 2B.

Example 3

Compositions with DPPA ratios in excess of DPPC and no divalent cationswere prepared. Dry lipids were weighed out and hydrated in dH₂ O byheating and stirring in the mole percentage ratio of from 15 to 25 mol %DPPC; from 65 to 75 mol % DPPA; from 0 to 20 mol % DPPE-PEG5,000. Asmall vial was rinsed with dH₂ O and tared on a Mettler AJ100 (MettlerInstrument Corp. PO Box 71, Hightstown, N.J., 08520) balance and five toten milligrams of the lyopholized lipid blend was weighed into the vial.Triple 0.22 μm filtered dI H₂ O was added by weight until the finalconcentration of lipid was 1 mg/ml. This mixture was heated to 45-50° C.for 1 hour and then sonicated in a Aquasonic cleaner Model 75 HT (VWR)at room temperature for 1 hour in 30 minute increments to preventexcessive heating. This treatment reduced the relative size of the lipidparticles in solution. The particles were sized using a NICOMP C370(Particle Sizing Systems, 75 Aero Camino Suite B, Santa Barbara Calif.93117) by a modified NNLS/CONTIN algorithm resulting in a multimodalsize distribution with peak intensity diameters as plotted in FIG. 4A.

Example 4

Compositions made with Ca²⁺ present in the original mixture wereprepared. The experiments in Example 3 were repeated except that 10 mMCaCl₂ was added to the original lipid mixtures. Sizing plots are shownin FIG. 4B.

Example 5

The experiments in Example 2 were repeated except that CaCl₂ was addedto the original lipid suspension rather than in the resuspension. FIG.3A shows the control size plots for the varying amounts of lipid andFIG. 3B shows the effect of the added Ca²⁺.

In Examples 6-10, DNA was added to variations of the lipid mixturesdescribed in the preceding Examples. The presence of DNA in the finalprecipitated lipid shows complexation. A control run without lipidsshowed no DNA in the final pellet. Ca²⁺ was added in Examples 8 and 9.

Example 6

Dry lipids were weighed out and hydrated in dI H₂ O by heating andstirring in the mole percentage ratio of 75 mol % DMPC to 25 mol % DMPAand then lyopholized. A small vial was rinsed with dI H₂ 0 and tared ona Mettler AJ100 (Mettler Instrument Corp. PO Box 71, Hightstown, N.J.,08520) balance and five to ten milligrams of the lyopholized lipid blendwas weighed into the vial. Triple 0.22 μm filtered dI H₂ O was added byweight until the final concentration of lipid was 1 mg/ml. This mixturewas heated to 45-50° C. for 1 hour and then sonicated in a Aquasoniccleaner Model 75 HT (VWR) at room temperature for 1 hour in 30 minuteincrements to prevent excessive heating.

200 μg of pCAT Control DNA (Promega, Madison, Wis.) was precipitatedwith CaCl₂ and ethanol. The dried DNA pellet was resuspended in 1milliliter of the 1 mg/ml lipid suspension and allowed to incubate atroom temperature for 1 hour. The lipid-DNA complex was then collected bycentrifugation at 12K rpm in an Eppendorf centrifuge 5415C(BrinkmanInstr. Inc., Westbury, N.Y. 11590). The supernatantwas removedto another tube and the pelleted lipid-DNA complex was resuspended in aminimal volume of dI H₂ O. The supernatant and the pellet sultions wereassayed for DNA using a Hoefer TKO 100 Fluorometer (Hoefer ScientificInstruments, San Francisco, Calif. 94117). The DNA concentration in thesupernatant was 5 μg/μl and the DNA concentration in the lipid-DNApelleted complex was 1572 μg/μl, indicating that the DNA was complexedwith the lipid.

Example 7

Dry lipids were weighed out and hydrated in dI H₂ O by heating andstirring in the mole percentage ratio of 25 mol % DMPC to 75 mol % DMPAand then lyopholized. A small vial was rinsed with dI H₂ O and tared ona Mettler AJ100 (Mettler Instrument Corp. PO Box 71, Hightstown, N.J.,08520) balance and five to ten milligrams of the lyopholized lipid blendwas weighed into the vial. Triple 0.22 μm filtered dI H₂ O was added byweight until the final concentration of lipid was 1 mg/ml. This mixturewas heated to 45-50° C. for 1 hour and then sonicated in a Aquasoniccleaner Model 75 HT (VWR) at room temperature for 1 hour in 30 minuteincrements to prevent excessive heating.

200 μg of pCAT Control DNA (Promega, Madison, Wis.) was precipitatedwith CaCl₂ and ethanol. The dried DNA pellet was resuspended in 1milliliter of the 1 mg/ml lipid suspension and allowed to incubate atroom temperature for 1 hour. The lipid-DNA complex was then collected bycentrifugation at 12K rpm in an Eppendorf centrifuge 5415C (BrinkmanInstr. Inc., Westbury, N.Y. 11590). The supernatant was removed toanother tube and the pelleted lipid-DNA complex was resuspended in aminimal volume of dl H₂ O. The supernatant and the pellet sultions wereassayed for DNA using a Hoefer TKO 100 Fluorometer (Hoefer ScientificInstruments, San Francisco, Calif. 94117). The DNA concentration in thesupernatant was 0 μg/μl and the DNA concentration in the lipid-DNApelleted complex was 67 μg/μl, indicating that the DNA was complexedwith the lipid.

Example 8

Dry lipids were weighed out and hydrated in dI H₂ O by heating andstirring in the mole percentage ratio of 70 mol % DMPC to 20 mol % DMPAto 10 mol % DMPE-PEG5,000, and then lyophilized. A small vial was rinsedwith dI H2O and tared on a Mettler AJ100 (Mettler Instrument Corp. POBox 71, Hightstown, N.J., 08520) balance and five to ten milligrams ofthe lyopholized lipid blend was weighed into the vial. Triple 0.22 μmfiltered dl H₂ O was added by weight until the final concentration oflipid was 1 mg/ml. This mixture was heated to 45-50° C. for 1 hour andthen sonicated in a Aquasonic cleaner Model 75 HT (VWR) at roomtemperature for 1 hour in 30 minute increments to prevent excessiveheating.

200 μg of pCAT Control DNA (Promega, Madison, Wis.) was precipitatedwith CaCl₂ and ethanol. The dried DNA pellet was resuspended in 1milliliter of the 1 mg/ml lipid suspension and allowed to incubate atroom temperature for 1 hour. The lipid-DNA complex was then collected bycentrifugation at 12K rpm in an Eppendorf centrifuge 5415C (BrinkmanInstr. Inc., Westbury, N.Y. 11590). The supernatant was removed toanother tube and the pelleted lipid-DNA complex was resuspended in aminimal volume of dl H₂ O. The supernatant and the pellet sultions wereassayed for DNA using a Hoefer TKO 100 Fluorometer (Hoefer ScientificInstruments, San Francisco, Calif. 94117). The DNA concentration in thesupernatant was 43 μg/μl and the DNA concentration in the lipid-DNApelleted complex was 1791 μg/μl, indicating that the DNA was complexedwith the lipid.

Example 9

Dry lipids were weighed out and hydrated in dI H₂ O by heating andstirring in the mole percentage ratio of 20 mol % DMPC to 70 mol % DMPAto 10 mol % DMPE-PEG5000, and then lyophilized. A small vial was rinsedwith dI H₂ O and tared on a Mettler AJ100 (Mettler Instrument Corp. POBox 71, Hightstown, N.J., 08520) balance and five to ten milligrams ofthe lyopholized lipid blend was weighed into the vial. Triple 0.22 μmfiltered dl H₂ O was added by weight until the final concentration oflipid was 1 mg/ml. This mixture was heated to 45-50C. for 1 hour andthen sonicated in a Aquasonic cleaner Model 75 HT (VWR) at roomtemperature for 1 hour in 30 minute increments to prevent excessiveheating.

200 μg of pCAT Control DNA (Promega, Madison, Wis.) was precipitatedwith CaCl₂ and ethanol. The dried DNA pellet was resuspended in 1milliliter of the 1 mg/ml lipid suspension and allowed to incubate atroom temperature for 1 hour. The lipid-DNA complex was then collected bycentrifugation at 12K rpm in an Eppendorf centrifuge 5415C (BrinkmanInstr. Inc., Westbury, N.Y. 11590). The supernatant was removed toanother tube and the pelleted lipid-DNA complex was resuspended in aminimal volume of dI H₂ O. The supernatant and the pellet sultions wereassayed for DNA using a Hoefer TKO 100 Fluorometer (Hoefer ScientificInstruments, San Francisco, Calif. 94117). The DNA concentration in thesupernatant was 28 μg/μl and the DNA concentration in the lipid-DNApelleted complex was 6800 μg/μl, indicating that the DNA was complexedwith the lipid.

Example 10

Dry lipids were weighed out and hydrated in dI H₂ O by heating andstirring in the mole percentage ratio of 20 mol % DMPC to 70 mol % DMPAto 10 mol % DMPE-PEG5000. A small vial was rinsed with dl H₂ O and taredon a Mettler AJ100 (Mettler Instrument Corp. PO Box 71, Hightstown,N.J., 08520) balance and five to ten milligrams of the lyopholized lipidblend was weighed into the vial. Triple 0.22 μm filtered dI H₂ O wasadded by weight until the final concentration of lipid was 1 mg/ml. Thismixture was heated to 45-50° C. for 1 hour and then sonicated in aAquasonic cleaner Model 75 HT (VWR) at room temperature for 1 hour in 30minute increments to prevent excessive heating.

200 μg of pCAT Control DNA (Promega, Madison, Wis.) was precipitatedwith CaCl₂ and ethanol. The dried DNA pellet was resuspended in 1milliliter of the 1 mg/ml lipid suspension and allowed to incubate atroom temperature for 1 hour. The lipid-DNA complex was then collected bycentrifugation at 12K rpm in an Eppendorf centrifuge 5415C (BrinkmanInstr. Inc., Westbury, N.Y. 11590). The supernatant was removed toanother tube and the pelleted lipid-DNA complex was resuspended in aminimal volume of dl H₂ O. The supernatant and the pellet sultions wereassayed for DNA using a Hoefer TKO 100 Fluorometer (Hoefer ScientificInstruments, San Francisco, Calif. 94117). The DNA recovered in thesupernatant was 183 μg and the total DNA in the pellet was 14.1 μg,indicating essentially no complexation of DNA with lipid in the absenceof calcium.

In Examples 11 and 12, dexamethasone was added to selected compositionsfrom the preceding Examples to determine the level of entrappment.

Example 11

Dexamethasone is a highly potent hydrophobic drug that is soluble at 100mg/L in water. A mixture was weighed out in the ratio of 65 mol % DPPAto 15 mol % DPPC to 20 mol % dexamethasone. This mixture was dissolvedin methanol and rotary evaporated under vacuum until it was a dry film.The film was subjected to hard vacuum overnight. The film wasresuspended in deionized water at 1 mg/ml and sonicated for 5 minutes.The resulting suspension was homogeneous. 100 mM CaCl₂ was addeddropwise while sonicating the suspension to a final concentration of 10mM. This resulted in the aggregation of the lipids and their subsequentprecipitation from the solution. The cochleates were sonicated for onehalf hour at 90 watts with no change in characteristics. The resultingcompositions were too large and nonhomogeneous without thepolymer-bearing lipid.

Example 12

Dexamethasone is soluble at 100 mg/L in water. A mixture was created byadding 80 mg of the lipid blend to 20 mg of dexamethasone. The lipidblend was 70 mol % DMPA, 20 mol % DMPC and 10 mol % DMPE-PEG5000. Theblend and the dexamethasone were dissolved in methanol and rotaryevaporated under vacuum until it was a dry film. The film was subjectedto hard vacuum overnight. The film was resuspended in deionized water at10 mg/ml and sonicated for 15 minutes at 90 watts. The resultingsuspension was homogeneous. 100 mM CaCl₂ was added dropwise whilesonicating the suspension to a final concentration of 10 mM. There wasno visible change it the solution characteristics after the addition ofthe cacium chloride solution which is consistent with prior resultsshowing no real change in size of the particles by QELS. One milliliterof this mixture was administered to a Sephacryl S-200-HR column 1/2" by7" running in deionized water at 1 ml/minute, collecting 3 ml fractions.The column was charaterised with a mixture of a fluoresent lipid blendof DPPE and DPPC and bromophenol blue, a visible dye. The lipid wasseparated from the dye. The lipid eluted in the 3rd and 4th fractionsand the dye began in the 10th fraction and was strongest in the 12ththrough the 17th fractions. Fractions 3 and 4 from the mixture ofdexamethasone and lipid blend were translucent with suspended lipid. Allof the remaining 80 fractions were clear. The fractions were frozen inliquid nitrogen and lyopholized. The lyophilized fractions weredissolved or resuspended in 5 mls of methanol and scanned at 235 nm inthe UV spectrophotometer. The absorbance maximum for dexamethasone inmethanol was 235-238 nm as determined by dissolving dexamethasone inmethanol and scanning from 320 nm through 220 nm. Pure methanol wasscanned between 320 nm and 190 nm and was found to have no absorbancebelow 210 nm. All samples were zeroed on pure methanol before scanningto prevent any carryover between samples. A standard curve wasconstructed from dexamethasone in methanol at 237 nm peak absorbance.The standard curve was between 2.5 and 25 ug/ml. The fractions thatcontained lipid were suspensions and could not be scanned accurately.The remaining fractions were scanned and presumably contained the free,unentrapped dexamethasone. The majority of the dexamethasone absorbancewas in fractions 11 through 15. The entire recovered free dexamethasonewas only 7.3 μg. 2 mgs were loaded on to the column in the 1 ml aliquotof the mixture which was 10 mg/ml, 20% of which was dexamethasone. Theexperiment suggested that the compositions of the present invention mayachieve high payloads of dexamethasone.

Example 13

The lipid preparation of Example 2 in 10 mM CaCl₂ is lyophilizedimmediately after the sonication step. A cryoprotectant, such asmannitol, glycerol, sorbitol or trehalose, is added to 1.0 mg/ml. Afterlyophilization, the powdered sample is stored or resuspended in dH₂ O.

Example 14

The lipid preparation of Example 2 in 10 mM CaCl₂ is lyophilizedimmediately after the sonication step. The sample contains PEG-2000 (2:1w/w) over the weight of the lipid mixture as a substitute cryoprotectantfor the sugars and sugar alcohols in Example 13. After lyophilization,the powdered sample is stored or resuspended in dH₂ O.

Example 15

Lyophilized compositions may be stored under a head space of apreselected gas, as described herein. The compositions from Example 14were placed in a sealed vessel and the head space of the air wasevacuated and replaced with perfluoropropane. The result was a pluralityof microvoids within and on the surface of the compositions filled byperfluoropropane gas. When the compositions were rehydrated, acousticanalysis showed the particles to be acoustically active. Optimalacoustic attenuation was achieved when the mixture was gently handshaken during reconstitution with saline rather than mixed in an ESPECapmix.

Example 16

A lipid blend of 82 mole % dipalmitoylphosphatidylcholine, 8 mole %dipalmitoylphophatidic acid, 10 mole %diplmitoylphosphatidylethanolamine-PEG 5000 was prepared with CaCl₂ asin Example 2. Perfluorohexane was added to the mixture at aconcentration of 10 mg/ml. This material was then extruded through anextrusion device (Lipex Biomembranes, Vancouver, B.C. Canada). Theextrusion was accomplished under nitrogen pressure at 1000 psi. Firstthe material was passed through 2 μm filter, then through 1 μm filterand then through a 0.4 μm filter five times. The material was then sizedby a quasi-elastic light scattering device (Particle Sizing Systems,Santa Barbara, Calif.) mean diameter of the particles was about 200-500nm.

Example 17

A lipid blend of 82 mole % dipalmitoylphosphatidylcholine, 8 mole %dipalmitoylphophatidic acid, 10 mole %diplmitoylphosphatidylethanolamine-PEG 5000 was prepared with CaCl₂ asin Example 2. Perfluoropentane was added to the mixture at aconcentration of 10 mg/ml. This material was then extruded through anextrusion device (Lipex Biomembranes, Vancouver, B.C. Canada). Theextrusion was accomplished under nitrogen pressure at 1000 psi. Firstthe material was passed through 2 μm filter, then through 1 μm filterand then through a 0.4 μm filter five times. The material was then sizedby a quasi-elastic light scattering device (Particle Sizing Systems,Santa Barbara, Calif.) mean diameter of the particles was about 200-500nm.

Example 18

A volatile organic solvent, preferably a perfluorocarbon, mostpreferably 1-bromoperfluorobutane, is incorporated into the core of thecompositions before the aggregation process (see Example 1). Thecompositions are then lyophilized, resulting in porous, solidstructures. These are stored under a head space of air or an insolublegas such as perfluoropropane or sulfur hexafluoride. The resultingporous gas-filled compositions are acoustically active. The drugs listedin the disclosure of the present invention can be incorporated into thecompositions for delivery with or without ultrasound according to themethods described above.

The disclosure of each patent, patent application and publication citedor described in this document is hereby incorporated by reference hereinin its entirety.

Various modifications of the invention, in addition to those describedherein, will be apparent to one skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thespirit and scope of the appended claims.

What is claimed is:
 1. A contrast agent comprising a cochleate whichcomprises a charged lipid, a counter ion, a lipid covalently bonded to apolymer, and a gas, a gaseous precursor or a gas and a gaseousprecursor, wherein the gas and gaseous precursor comprise fluorinatedcompounds.
 2. The contrast agent of claim 1, wherein the charged lipidis an anionic lipid and the counter ion is a cationic counter ion. 3.The contrast agent of claim 2, wherein the anionic lipid is selectedfrom the group consisting of a phosphatidic acid, a phosphatidylglycerol, a phosphatidyl glycerol fatty acid ester, a phosphatidylethanolamine anandamide, a phosphatidyl ethanolamine methanandamide, aphosphatidyl serine, a phosphatidyl inositol, a phosphatidyl inositolfatty acid ester, a cardiolipin, a phosphatidyl ethylene glycol, anacidic lysolipid, a sulfolipid, a sulfatide, a saturated free fattyacid, an unsaturated free fatty acid, a palmitic acid, a stearic acid,an arachidonic acid, an oleic acid, a linolenic acid, a linoleic acid,and a myristic acid.
 4. The contrast agent of claim 2, wherein thecationic counter ion is selected from the group consisting of Be²⁺,Mg²⁺, Ca²⁺, Sr2+, Ba²⁺, Al³⁺, Ga³⁺, Ge³⁺, Sn⁴⁺, Pb²⁺, Pb⁴⁺, Ti³⁺, Ti⁴⁺,V²⁺, V³⁺, Cr²⁺, Cr³⁺, Mn²⁺, Mn³⁺, Fe²⁺, Fe³⁺, Co²⁺, Co³⁺, Ni²⁺, Ni³⁺,Cu²⁺, Zn²⁺, Zr⁴⁺, Nb³⁺, Mo²⁺, Mo³⁺, Cd²⁺, In³⁺, W²⁺, W⁴⁺, Os²⁺, Os³⁺,Os⁴⁺, Ir²⁺, Ir³⁺, Ir⁴⁺, Hg²⁺, Bi³⁺, La³⁺, and Gd³⁺.
 5. The contrastagent of claim 4, wherein the cationic counter ion is selected from thegroup consisting of Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺ and Gd³⁺.
 6. The contrastagent of claim 5, wherein the cationic counter ion is Ca²⁺.
 7. Thecontrast agent of claim 1, wherein, in the lipid covalently bonded tothe polymer, the polymer is selected from the group consisting ofpolyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol,polypropylene glycol, a polyvinylalkylether, a polyacrylamide, apolyalkyloxazoline, a polyhydroxyalkyloxazoline, a polyphosphazene, apolyoxazolidine, a polyaspartamide, a polymer of sialic acid, apolyhydroxyalkyl(meth)acrylate and a poly(hydroxyalkylcarboyxlic acid).8. The contrast agent of claim 7, wherein, in the lipid covalentlybonded to the polymer, the polymer is polyethylene glycol.
 9. Thecontrast agent of claim 8, wherein the polyethylene glycol has amolecular weight of from about 1,000 to about 10,000.
 10. The contrastagent of claim 1, wherein the lipid covalently bonded to the polymer isselected from the group consistingofdipalmitoylphosphatidylethanolamine-polyethylene glycol,dioleoylphosphatidylethanolamine-polyethylene glycol anddistearylphosphatidylethanolamine-polyethylene glycol.
 11. The contrastagent of claim 2, wherein the anionic lipid is dipalmitoyl-phosphatidicacid, the cationic counter ion is Ca²⁺ and the lipid covalently bondedto the polymer is dipalmitoylphosphatidylethanolamine-polyethyleneglycol.
 12. The contrast agent of claim 1, further comprising at leastone lipid having a neutral charge.
 13. The contrast agent of claim 1,further comprising a targeting ligand.
 14. The contrast agent of claim13, wherein the targeting ligand is selected from the group consistingof peptides, proteins and saccharides.
 15. The contrast agent of claim1, wherein the fluorinated compound is selected from the groupconsisting of a perflurocarbon, sulfur hexafluoride and aperfluoroether.
 16. The contrast agent of claim 15, wherein thefluorinated compound is a perfluorocarbon selected from the groupconsisting of perfluoromethane, perfluoroethane, perfluoropropane,perfluorocyclopropane, perfluorobutane, perfluorocyclobutane,perfluoropentane and perfluorocyclopentane.
 17. The contrast agent ofclaim 15, wherein the fluorinated compound is a perfluoroether selectedfrom the group consisting of perfluorotetrahydropyran,perfluoromethyltetrahydrofuran, perfluorobutylmethyl ether,perfluoropropylethyl ether, perfluorocyclobutylmethyl ether,perfluorocyclopropylethyl ether, perfluoropropylmethyl ether,perfluorodiethyl ether, perfluorocyclopropylmethyl ether,perfluoromethylethyl ether and perfluorodimethyl ether.
 18. The contrastagent of claim 1, further comprising a fluorinated liquid.
 19. Thecontrast agent of claim 18, wherein the fluorinated liquid is selectedfrom the group consisting of perfluorohexane, perfluoroheptane,perfluorooctane, perfluorononane, perfluorodecane, perfluorododecane,perfluorocyclohexane, perfluorodecalin, perfluorododecalin,perfluorooctyliodide, perfluorooctylbromide, perfluorotripropylamine,perfluorotributylamine, perfluorobutylethyl ether,bis(perfluoroisopropyl) ether and bis(perfluoropropyl) ether.
 20. Acontrast agent of claim 1 wherein said cochleate is in the form of aspiral or tubule.